U.S. patent number 10,072,092 [Application Number 14/620,955] was granted by the patent office on 2018-09-11 for methods of use of anti-cd19 antibodies with reduced immunogenicity.
This patent grant is currently assigned to Merck Patent GmbH. The grantee listed for this patent is Merck Patent GmbH. Invention is credited to Jonathan Davis, Pascal Andre Stein, Michael Super.
United States Patent |
10,072,092 |
Super , et al. |
September 11, 2018 |
**Please see images for:
( Certificate of Correction ) ** |
Methods of use of anti-CD19 antibodies with reduced
immunogenicity
Abstract
Anti-CD19 B4 antibodies with modified variable regions are
disclosed. The modified anti-CD19 variable region polypeptides have
alterations to one or more framework regions or complementarity
determining regions of the heavy chain variable region or light
chain variable region, thereby to reduce a T-cell response.
Inventors: |
Super; Michael (Lexington,
MA), Davis; Jonathan (Auburndale, MA), Stein; Pascal
Andre (Boston, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Merck Patent GmbH |
Darmstadt |
N/A |
DE |
|
|
Assignee: |
Merck Patent GmbH (Darmstadt,
DE)
|
Family
ID: |
37865674 |
Appl.
No.: |
14/620,955 |
Filed: |
February 12, 2015 |
Prior Publication Data
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Document
Identifier |
Publication Date |
|
US 20150322159 A1 |
Nov 12, 2015 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14222517 |
Mar 21, 2014 |
8957195 |
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11648505 |
Apr 8, 2014 |
8691952 |
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60755609 |
Dec 30, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
25/00 (20180101); A61P 37/04 (20180101); A61P
37/02 (20180101); A61P 21/04 (20180101); A61P
37/06 (20180101); C07K 16/30 (20130101); A61P
1/04 (20180101); A61P 19/02 (20180101); C07K
16/2896 (20130101); C07K 16/2803 (20130101); C07K
16/2866 (20130101); A61K 39/3955 (20130101); A61P
35/00 (20180101); A61P 29/00 (20180101); A61K
39/3955 (20130101); A61K 2300/00 (20130101); C07K
2317/24 (20130101); C07K 2317/41 (20130101); A61K
2039/505 (20130101); C07K 2317/56 (20130101); C07K
2317/567 (20130101); C07K 2317/732 (20130101) |
Current International
Class: |
A61K
39/395 (20060101); C07K 16/28 (20060101); A61K
39/40 (20060101); C07K 16/30 (20060101); C07K
16/00 (20060101); A61K 39/00 (20060101) |
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Primary Examiner: Dahle; Chun Wu
Attorney, Agent or Firm: Goodwin Procter LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of U.S. patent
application Ser. No. 14/222,517, filed Mar. 21, 2014, which is a
divisional application of U.S. patent application Ser. No.
11/648,505, filed Dec. 29, 2006, which claims the benefit of and
priority to U.S. Provisional Patent Application No. 60/755,609,
filed Dec. 30, 2005, the entire disclosures of each of which are
incorporated herein by reference.
Claims
What is claimed is:
1. A method of treating a patient having B-cell lymphoma or an
autoimmune disease, the method comprising the step of administering
to a patient a therapeutically effective amount of an anti-CD19
antibody comprising a variable domain comprising: a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO:13
with an amino acid substitution at one or more residues
corresponding to Gln5, Arg19, Leu20, Arg40, Gln43, Lys65, Ser85,
Ser88, and Val93; and a light chain variable region comprising the
amino acid sequence of SEQ ID NO:25 with an amino acid substitution
at one or more residues corresponding to Ile10, Met11, Val19,
Ser51, and Leu53.
2. The method of claim 1, wherein the heavy chain variable region
comprises one or more substitutions selected from Gln5Glu,
Arg19Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp,
Ser88Ala, and Val93Thr.
3. The method of claim 1, wherein the heavy chain variable region
comprises the amino acid sequence of SEQ ID NO:17.
4. A method for targeting a cell with CD19 on its surface, the
method comprising the step of administering an anti-CD19 antibody
comprising a variable domain comprising: a heavy chain variable
region comprising the amino acid sequence of SEQ ID NO:13 with an
amino acid substitution at one or more residues corresponding to
Gln5, Arg19, Leu20, Arg40, Gln43, Lys65, Ser85, Ser88, and Val93;
and a light chain variable region comprising the amino acid
sequence of SEQ ID NO:25 with an amino acid substitution at one or
more residues corresponding to Ile10, Met11, Val19, Ser51, and
Leu53.
5. The method of claim 4, wherein the heavy chain variable region
comprises one or more substitutions selected from Gln5Glu,
Arg19Lys, Leu20Val, Arg40Thr, Gln43Lys, Lys65Asp, Ser85Asp,
Ser88Ala, and Val93Thr.
6. The method of claim 4, wherein the light chain variable region
comprises one or more substitutions selected from Ile10Thr,
Met11Leu, Val19Ala, Ser51Asp, and Leu53Thr.
7. The method of claim 4, wherein the heavy chain variable region
comprises the amino acid sequence of SEQ ID NO:17.
8. The method of claim 1, wherein the patient has B-cell
lymphoma.
9. The method of claim 4, wherein the cell is a tumor cell.
10. The method of claim 9, wherein the tumor cell is a B-cell
lymphoma tumor cell.
11. The method of claim 1, wherein the light chain variable region
comprises one or more substitutions selected from Ile10Thr,
Met11Leu, Val19Ala, Ser51Asp, and Leu53Thr.
12. The method of claim 1, wherein the light chain variable region
comprises the amino acid sequence of SEQ ID NO:29.
13. The method of claim 4, wherein the light chain variable region
comprises the amino acid sequence of SEQ ID NO:29.
14. The method of claim 1, wherein the heavy chain variable region
comprises the amino acid sequence of SEQ ID NO:17 and the light
chain variable region comprises the amino acid sequence of SEQ ID
NO:29.
15. The method of claim 4, wherein the heavy chain variable region
comprises the amino acid sequence of SEQ ID NO:17 and the light
chain variable region comprises the amino acid sequence of SEQ ID
NO:29.
16. The method of claim 8, wherein the heavy chain variable region
comprises the amino acid sequence of SEQ ID NO:17 and the light
chain variable region comprises the amino acid sequence of SEQ ID
NO:29.
17. The method of claim 10, wherein the heavy chain variable region
comprises the amino acid sequence of SEQ ID NO:17 and the light
chain variable region comprises the amino acid sequence of SEQ ID
NO:29.
Description
FIELD OF THE INVENTION
The invention relates generally to variable regions of the
anti-CD19 murine monoclonal antibody B4 light chain and heavy chain
modified to reduce their immunogenicity.
BACKGROUND
CD19 is a surface protein found on B cells and on certain cancerous
cells derived from B cells, such as many B cell lymphomas.
Anti-CD19 monoclonal antibodies have been generated in mice, and
show some promise in pre-clinical animal models of B cell-derived
cancers. However, mouse-derived antibodies are generally
immunogenic in humans. A number of strategies have been developed
to alter mouse-derived antibodies to minimize their immunogenicity
in humans. One such strategy, chimerization, involves the fusion of
mouse variable/regions to human constant regions. However, the
mouse-derived variable region sequences remaining following
chimerization will often be immunogenic. Another such strategy,
humanization, involves the replacement of mouse-derived framework
regions (FRs) within the variable regions with the most closely
related human-derived sequences, with the optional reversion of
certain amino acids back to the corresponding mouse amino acid in
order to maintain binding activity. However, even humanized
antibodies may be immunogenic, since the antibody complementarity
determining regions (CDRs) generally contain B cell epitopes and T
cell epitopes that are non-self. Indeed, even fully human
antibodies are immunogenic; this is the basis for the formation of
anti-idiotype antibodies during the course of an immune response.
All of these problems may apply to mouse-derived anti-CD19
antibodies as they would to any other type of antibody. Therefore,
there is a need for anti-CD19 antibodies with reduced
immunogenicity.
SUMMARY OF THE INVENTION
The present invention is directed to an anti-CD19 murine monoclonal
B4 antibody which has been modified to reduce its immunogenicity in
comparison to wild-type B4 antibody. More specifically, the
variable region of the B4 antibody of the invention is modified to
remove potential T-cell epitopes. As a result, B4 antibodies of the
invention have improved biological properties compared to wild-type
B4 antibodies.
Accordingly, in one aspect, the invention features an amino acid
sequence defining a modified immunoglobulin heavy chain framework
region comprising amino acid residues 1-30 of SEQ ID NO:22, wherein
one or more of the amino acid residues at positions X5, X12, X19,
X20, X23 and X24 are as follows: X5 is Q or E, X12 is V or K, X19
is R or K, X20 is L or V, X23 is K, E or D, or X24 is T or A.
According to this aspect of the invention, at least one of the
amino acid residues at positions X5, X12, X19, X20, X23, or X24 is
not the same amino acid residue as the amino acid at the
corresponding position in the unmodified immunoglobulin heavy chain
framework region as set forth in amino acid residues 1-30 of SEQ ID
NO:13. In one embodiment, X23 is E or D.
In another aspect, the invention features an amino acid sequence
defining a modified immunoglobulin heavy chain framework region
comprising amino acid residues 1-14 of SEQ ID NO:23, wherein one or
more of the amino acid residues at positions X3, X5, X7, and X8,
are as follows: X3 is K or R, X5 is R, T, or A, X7 is G, D, or E,
or X8 is Q or K. According to this aspect of the invention, at
least one of the amino acid residues at positions X3, X5, X7, or X8
is not the same as the amino acid at the corresponding position in
the unmodified immunoglobulin heavy chain framework region as set
forth in amino acid residues 36-49 of SEQ ID NO:13. In one
embodiment, X7 is E or D.
In another aspect, the invention features an amino acid sequence
defining a modified immunoglobulin heavy chain framework region
comprising amino acid residues 1-39 of SEQ ID NO:24, wherein one or
more of the amino acid residues at positions X6, X10, X26, X29, and
X34 are as follows: X6 is K, D, or E, X10 is K, E, or D, X26 is S,
D, or E, X29 is S or A, or X34 is V or T. According to this aspect
of the invention, at least one of the amino acid residues at
positions X6, X10, X26, X29, or X34 is not the same as the amino
acid at the corresponding position in the unmodified immunoglobulin
heavy chain framework region as set forth in amino acid residues
60-98 of SEQ ID NO:13. In one embodiment, X10 is E or D.
According to another aspect, the invention features an amino acid
sequence defining a modified immunoglobulin light chain framework
region comprising amino acid residues 1-23 of SEQ ID NO:32, wherein
one or more of the amino acid residues at positions X1, X3, X7,
X10, X11, and X19 are as follows: X1 is Q or D, X3 is V or A, X7 is
S or E, X10 is I or T, X11 is M or L, or X19 is V or A. According
to this aspect of the invention, at least one of the amino acid
residues at positions X1, X3, X7, X10, X11, or X19 is not the same
as the amino acid at the corresponding position in the unmodified
immunoglobulin light chain framework region as set forth in amino
acid residues 1-23 of SEQ ID NO:25. In one embodiment, X3 is A and
X7 is E. In another embodiment, X1 is D, X10 is I, and X11 is
L.
In another aspect, the invention features an amino acid sequence
defining a modified immunoglobulin light chain complementarity
determining region comprising amino acid residues 24-33 of SEQ ID
NO:28.
In another aspect, the invention features an amino acid sequence
defining a modified immunoglobulin light chain framework region
comprising amino acid residues 56-87 of SEQ ID NO:28.
According to another aspect, the invention features an antibody
variable region comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,
SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID
NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO:
31, wherein the antibody variable region specifically binds to
CD19.
According to another aspect, the invention features a polypeptide
at least 90% or at least 95% identical to a B4 antibody heavy chain
variable region, the polypeptide comprising an amino acid
substitution at one or more residues corresponding to Val12, Leu20,
Lys23, Thr24, Lys38, Gly42, Gln43, Lys65, Lys69, Ser85, Ser88, or
Val93. In one embodiment, the polypeptide comprises one or more of
substitutions Gln5Glu, Val12Lys, Arg19Lys, Leu20Val, Lys23Glu,
Lys23Asp, Thr24Ala, Lys38Arg, Arg40Thr, Gly42Asp, Gly42Glu,
Gln43Lys, Lys65Asp, Lys65Glu, Lys69Glu, Lys69Asp, Ser85Asp,
Ser85Glu, Ser88Ala, or Val93Thr.
According to another aspect, the invention features a polypeptide
at least 90% or at least 95% identical to a B4 antibody light chain
variable region, the polypeptide comprising an amino acid
substitution at one or more residues corresponding to Val3, Ser7,
Ile10, Met11, Val19, Val29, Ser51, Leu53, Ala54, or Ser75. In one
embodiment, the polypeptide comprises one or more of substitutions
Gln1Asp, Val3Ala, Ser7Glu, Ile10Thr, Met11Leu, Val19Ala, Val29Ala,
Ser51Asp, Leu53Thr, Ala54Asp, or Ser75Glu.
In another aspect, the invention features a nucleic acid encoding a
polypeptide according to arty one of the embodiments of the
invention.
In another aspect, the invention features a method of treating a
patient, the method comprising the step of administering a
therapeutically effective amount of a polypeptide according to any
one of the embodiments of the invention to a patient.
In another aspect, the invention features a method for targeting a
cell with CD19 on its surface, the method comprising the step of
administering an antibody variable region according to any one of
the embodiments of the invention. In one embodiment of the method
the cell is a tumor cell.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the nucleic acid sequence encoding B4 antibody heavy
chain variable region (B4 VH0) (SEQ ID NO:1).
FIG. 2 depicts the nucleic acid sequence encoding an exemplary B4
antibody heavy chain variable region incorporating codons for the
mutations K23E, G42D, K69E, and S85D (B4 VHv1) (SEQ ID NO:2).
FIG. 3 depicts the nucleic acid sequence encoding an exemplary B4
antibody heavy chain variable region incorporating codons for the
mutations K69E, and S85D (B4 VHv2) (SEQ ID NO:3).
FIG. 4 depicts the nucleic acid sequence encoding an exemplary B4
antibody heavy chain variable region incorporating codons for the
mutations Q5E, V12K, R19K, L20V, T24A, S85D, and S88A (B4 VHv3)
(SEQ ID NO:4).
FIG. 5 depicts the nucleic acid sequence encoding an exemplary B4
antibody heavy chain variable region incorporating codons for the
mutations Q5E, R19K, L20V, R40T, Q43K, K65D, S85D, S88A, and V93T
(B4 VHv4) (SEQ ID NO:5).
FIG. 6 depicts the nucleic acid sequence encoding an exemplary B4
antibody heavy chain variable region incorporating codons for the
mutations Q5E, V12K, R19K, L20V, T24A, K38R, R40A, Q43K, K65D,
S85D, and V93T (B4 VHv5) (SEQ ID NO:6).
FIG. 7 depicts the nucleic acid sequence encoding an exemplary B4
antibody heavy chain variable region incorporating codons for the
mutations Q5E, R19K, L20V, K65D, S85D, and V93T (B4 VHv6) (SEQ ID
NO:7).
FIG. 8 depicts the nucleic acid sequence encoding B4 antibody light
chain variable region (B4 VK0) (SEQ ID NO:8).
FIG. 9 depicts the nucleic acid sequence encoding an exemplary B4
antibody light chain variable region incorporating codons for the
mutations V3A, S7E, and A54D (B4 VKv1) (SEQ ID NO:9).
FIG. 10 depicts the nucleic acid sequence encoding an exemplary B4
antibody light chain variable region incorporating codons for the
mutations Q1D, I10T, M11L, and A54D (B4 VKv2) (SEQ ID NO:10).
FIG. 11 depicts the nucleic acid sequence encoding an exemplary B4
antibody light chain variable region incorporating codons for the
mutations I10T, M11L, V19A, V29A, and S75E (B4 VKv3) (SEQ ID
NO:11).
FIG. 12 depicts the nucleic acid sequence encoding an exemplary B4
antibody light chain variable region incorporating codons for the
mutations I10T, M11L, V19A, S51D, and L53T (B4 VKv4) (SEQ ID
NO:12).
FIG. 13 depicts the amino acid sequence of B4 antibody heavy chain
variable region (B4 VH0) (SEQ ID NO:13).
FIG. 14 depicts the amino acid sequence of an exemplary B4 antibody
heavy chain variable region with the mutations K23E, G42D, K69E,
and S85D (B4 VHv1) (SEQ ID NO:14).
FIG. 15 depicts the amino acid sequence of an exemplary B4 antibody
heavy chain variable region with the mutations K69E, and S85D (B4
VHv2) (SEQ ID NO:15).
FIG. 16 depicts the amino acid sequence of an exemplary B4 antibody
heavy chain variable region with the mutations Q5E, V12K, R19K,
L20V, T24A, S85D, and S88A (B4 VHv3) (SEQ ID NO:16).
FIG. 17 depicts the amino acid sequence of an exemplary B4 antibody
heavy chain variable region with the mutations Q5E, R19K, L20V,
R40T, Q43K, K65D, S85D, S88A, and V93T (B4 VHv4) (SEQ ID
NO:17).
FIG. 18 depicts the amino acid sequence of an exemplary B4 antibody
heavy chain variable region with the mutations Q5E, V12K, R19K,
L20V, T24A, K38R, R40A, Q43K, K65D, S85D, and V93T (B4 VHv5) (SEQ
ID NO:18).
FIG. 19 depicts the amine acid sequence of an exemplary B4 antibody
heavy chain variable region with the mutations Q5E, R19K, L20V,
K65D, S85D, and V93T (B4 VHv6) (SEQ ID NO:19).
FIG. 20 depicts the amino acid sequence of an exemplary B4 antibody
heavy chain variable region with the mutations V12K, K23E, G42D,
K65D, K69E, and S85D (B4 VHv11) (SEQ ID NO:20).
FIG. 21 depicts the amino acid sequence of an exemplary B4 antibody
heavy chain variable region with the mutations Q5E, V12K, R19K,
L20V, T24A, R40T, Q43K, K65D, S85D, S88A, and V93T (B4 VHv34) (SEQ
ID NO:21).
FIG. 22 depicts the amino acid sequence of an exemplary B4 antibody
heavy chain framework region with undefined amino acid residues X5,
X12, X19, X20, X23, and X24 (B4 VHfr1) (SEQ ID NO:22).
FIG. 23 depicts the amino acid sequence of an exemplary B4 antibody
heavy chain framework region 2 with undefined amino acid residues
X3, X5, X7, and X8 (B4 VHfr2) (SEQ ID NO: 23)
FIG. 24 depicts the amino acid sequence of an exemplary B4 antibody
heavy chain framework region 3 with undefined amino acid residues
X6, X10, X26, X29, and X34 (B4 VHfr3) (SEQ ID NO:24).
FIG. 25 depicts the amino acid sequence of B4 antibody light chain
variable region (B4 VK0) (SEQ ID NO:25).
FIG. 26 depicts the amino acid sequence of an exemplary B4 antibody
light chain variable region with the mutations V3A, S7E, and A54D
(B4 VKv1) (SEQ ID NO:26).
FIG. 27 depicts the amino acid sequence of an exemplary B4 antibody
light chain variable region with the mutations Q1D, I10T, M11L, and
A54D (B4 VKv2) (SEQ ID NO:27).
FIG. 28 depicts the amino acid sequence of an exemplary B4 antibody
light chain variable region with the mutations I10T, M11L, V19A,
V29A, and S75E (B4 VKv3) (SEQ ID NO:28).
FIG. 29 depicts the amino acid sequence of an exemplary B4 antibody
light chain variable region with the mutations I10T, M11L, V19A,
S51D, and L53T (B4 VKv4) (SEQ ID NO:29).
FIG. 30 depicts the amino acid sequence of an exemplary B4 antibody
light chain variable region with the mutations V3A, S7E, V19A,
A54D, and S75E (B4 VKv11) (SEQ ID NO:30).
FIG. 31 depicts the amino acid sequence of an exemplary B4 antibody
light chain variable region with the mutations I10T, M11L, V19A,
V29A, S51D, L53T, and S75E (B4 VKv34) (SEQ ID NO:31).
FIG. 32 depicts the amino acid sequence of an exemplary B4 antibody
light chain framework region with undefined amino acid residues X1,
X3, X7, X10, X11, and X19 (B4 VKfr1) (SEQ ID NO:32).
FIG. 33 depicts the amino acid sequence of an exemplary B4 antibody
light chain complementarity determining region with undefined amino
acid residues X3, X5, and X6 (B4 VKcdr2) (SEQ ID NO:33).
FIG. 34 depicts the amino acid sequence of B4 antibody heavy chain
variable region. The complementarity determining regions are
underlined. The modifiable amino acid residues are shown in
bold.
FIG. 35 depicts the amino acid sequence of B4 antibody light chain
variable region. The complementarity determining regions are
underlined. The modifiable amino acid residues are shown in
bold.
FIG. 36 is an amino acid sequence alignment of B4 antibody heavy
chain variable regions VH0 (SEQ ID NO:13), VHv1 (SEQ ID NO:14),
VHv2 (SEQ ID NO:15), VHv3 (SEQ ID NO:16), VHv4 (SEQ ID NO:17), VHv5
(SEQ ID NO:18), VHv11 (SEQ ID NO:20), and VHv34 (SEQ ID NO:21).
FIG. 37 is an amino acid sequence alignment of B4 antibody light
chain variable regions VK0 (SEQ ID NO:25), VKv1 (SEQ ID NO:26),
VKv2 (SEQ ID NO:27), VKv3 (SEQ ID NO:28), VKv4 (SEQ ID NO:29),
VKv11 (SEQ ID NO:30), and VKv34 (SEQ ID NO:31).
FIG. 38 shows the results of an ADCC assay on Daudi Burkitt's
lymphoma cells performed with B4 VHv4/VKv4 antibody expressed
either from HEK 293T cells (empty triangles) or from YB2/0 cells
(filled triangles), and B4 VHv5/VKv4 antibody expressed either from
a NS/0 cell line (empty circles) or from YB2/0 cells (filled
circles) as described in Example 4.
FIG. 39 shows the results of treatment of mice transplanted with
human PBMCs treated with either the B4 VHv4/VKv4 antibody of the
invention (striped bars), Leu 16 antibody (white bars) or PBS
(black bars) as described in Example 5.
FIG. 40 shows the results of treatment of mice carrying Namalwa
lymphoma cells treated with either the B4 VHv4/VKv4 antibody of the
invention (empty circles) or PBS (stars) as described in Example
6
FIG. 41 shows the results of treatment of mice carrying Daudi
Burkitt's lymphoma cells treated with either the B4 VHv4/VKv4
antibody of the invention (empty triangles), cyclophosphamide
(empty squares), a combination of the B4 VHv4/VKv4 antibody and
cyclophosphamide (empty circles), or PBS (stars) as described in
Example 7.
FIGS. 42(a)-(c) show the results of treatment of mice carrying
Namalwa lymphoma cells with an antibody of the invention combined
with various chemotherapy agents, as described in Example 8. In
FIG. 42(a) treatments are cyclophosphamide (empty squares), the B4
VHv4/VKv4 antibody (X), a combination of the B4 VHv4/VKv4 antibody
and cyclophosphamide (filled squares), or PBS (stars). In FIG.
42(b) treatments are vincristine (empty triangles), the B4
VHv4/VKv4 antibody (X), a combination of the B4 VHv4/VKv4 antibody
and vincristine (filled triangles), or PBS (stars). In FIG. 42(c)
treatments are doxorubicin (empty circles), the B4 VHv4/VKv4
antibody (X), a combination of the B4 VHv4/VKv4 antibody and
doxorubicin (filled circles), or PBS (stars).
DETAILED DESCRIPTION OF THE INVENTION
The invention is directed to B4 proteins that have reduced
immunogenicity as compared to wild-type B4, as well as methods for
making and using such proteins. More specifically, the invention
provides mutations within a B4 antibody that have the effect of
reducing the immunogenicity of a B4 antibody itself, primarily by
removing T-cell epitopes within B4 that may stimulate to an immune
response.
The present invention is directed to a set of modified and body
heavy chain (VH) and light chain (VK) variable regions of the
anti-CD19 murine antibody B4 (Nadler et al., (1983) J. Immunol.
130:2947-2951; Roguska et al., (1994) Proc. Natl. Acad. Sci. USA
91:969-973), which herein are generically termed "B4 VHvx" and "B4
VKvy", respectively. For reference, the sequence of the heavy chain
variable region of the original murine B4 antibody (B4 VH0) and the
sequence of the light chain variable regions of the original murine
B4 antibody (B4 VK0) with the CDRs underlined, are provided in
FIGS. 34 and 35, respectively.
As compared to the original B4 VH0 and B4 VK0 polypeptides, B4 VHvx
and B4 VKvy polypeptides have reduced immunogenicity. More
specifically, the invention provides mutations within B4 VH and/or
B4 VK which have the effect of reducing the immunogenicity of B4
variable region polypeptides, primarily by removing T-cell epitopes
within these polypeptides that may stimulate an immune response.
According to the invention, protein compositions containing the
modified forms of the B4 variable regions are less immunogenic when
administered to a human, but are still competent to specifically
bind CD19 and to target cells expressing CD19.
As used herein, the terms "Complementarity-Determining Regions" and
"CDRs" are understood to mean the hypervariable regions or loops of
an immunoglobulin variable region that interact primarily with an
antigen. The immunoglobulin heavy chain variable region (VH) and
immunoglobulin light chain variable region (VK) both contain three
CDRs interposed between framework regions, as shown in FIGS. 34 and
35, respectively. For example, with reference to the amino acid
sequence defining the immunoglobulin heavy chain variable region of
the B4 antibody as shown in FIG. 34 (SEQ ID NO:13), the CDRs are
defined by the amino acid sequences from Ser31 to His35 (CDR1),
from Glu50 to Asn59 (CDR2), and from Gly99 to Tyr109 (CDR3). With
reference to the amino acid sequence defining the immunoglobulin
light chain variable region of the B4 antibody as shown in FIG. 35
(SEQ ID NO:25), the CDRs are defined by the amino acid sequences
from Ser24 to His33 (CDR1), from Asp49 to Ser55 (CDR2), and from
His88 to Thr94 (CDR3).
As used herein, the terms "Framework Regions" and "FRs" are
understood to mean the regions of an immunoglobulin variable region
adjacent to the Complementarity-Determining Regions. The
immunoglobulin heavy chain variable region (VH) and immunoglobulin
light chain variable region (VK) each contain four FRs, as shown in
FIGS. 34 and 35. For example, with reference to the amino acid
sequence defining the immunoglobulin heavy chain variable of the of
the B4 antibody as shown in FIG. 34 (SEQ ID NO: 13), the FRs are
defined by the amino acid sequences from Gln1 to Thr30 (FR1), from
Trp36 to Gly49 (FR2), from Tyr60 to Arg98 (FR3), and from Trp110 to
Ser120 (FR4). With reference to the amino acid sequence defining
the immunoglobulin light chain variable region of the B4 antibody
as shown in FIG. 35 (SEQ ID NO: 25), the FRs are defined by the
amino acid sequences from Gln1 to Cys23 (FR1), from Trp34 to Tyr48
(FR2), from Gly56 to Cys87 (FR3), and from Phe95 to Lys104 (FR4).
Furthermore, amino acid residues depicted in bold in FIGS. 34 and
35 are amino acid residues that may be mutated according to various
embodiments of the invention.
T-cell epitopes can be identified by a variety of computer and
non-computer methods, including predictions based on
structure-based computer modeling or by synthesis of peptides and
testing for binding to specific MHC Class II molecules in an
immunogenicity assay. According to the invention, a potential
T-cell epitope is a sequence that, when considered as an isolated
peptide, is predicted to bind to an MHC Class II molecule or an
equivalent in a non-human species. A potential T-cell epitope is
defined without consideration of other aspects of antigen
processing, such as the efficiency of protein uptake into
antigen-presenting cells, the efficiency of cleavage at sites in an
intact protein to yield a peptide that can bind to MHC Class II,
and so on. Thus, the set of T-cell epitopes that are actually
presented on MHC Class II after administration of a protein to an
animal is a subset of the potential T-cell epitopes. According to
the invention, a T-cell epitope is an epitope on a protein that
interacts with an MHC class II molecule. Without wishing to be
bound by theory, it is understood that a T-cell epitope is an amino
acid sequence in a protein that failed to undergo the negative
T-cell selection process during T-cell development and therefore
will be expected to be presented by an MHC Class II molecule and
recognized by a T-cell receptor.
According to one embodiment, the invention provides methods related
to reducing the immonogenicity of B4 VH and B4 VK regions.
According to one embodiment of the invention, potential non-self
T-cell epitopes are identified in sequences of B4 VH or B4 VK. For
example, potential non-self T-cell epitopes are identified by
computational methods based on modeling peptide binding to MHC
Class II molecules. Substitutions to specific amino acid residues
ape then made such that the ability of peptides containing
potential T-cell epitopes to bind to MHC Class II is reduced or
eliminated.
Modified Protein Sequences of Variable Regions of the
Invention.
According to one embodiment, the effect of a specific amino acid
mutation or mutations is predicted based on structure-based
computer modeling. For example, ProPred
(http://www.imtech.res.in/raghava/propred; Singh and Raghava (2001)
Bioinformatics 17:1236-1237) is a publically available web-based
tool that can be used for the prediction of peptides that bind
HLA-DR alleles, ProPred is based on a matrix prediction algorithm
described by Stumiolo for a set of 50 HLA-DR alleles (Stumiolo et
al., (1999) Nature Biotechnol. 17:555-561). Using such an
algorithm, various peptide sequences were discovered within B4 VH
and B4 VK which are predicted to bind to multiple MHC class II
alleles and are therefore likely to be immunogenic. These peptide
sequences and their predicted binding frequency to HLA-DR alleles
are shown in Table 1.
With reference to Table 1, the sequence of each 9-mer peptide that
binds to at least 5 HLA-DR alleles is indicated, along with its
position (#) in the B4 VH region (left column) or B4 VK region
(right column). "Bind freq." refers to the number of alleles, out
of a possible 50 alleles, that the peptide binds, above an
arbitrary binding threshold, in this case 20%. A binding frequency
of "+" indicates the peptide binds to 5-9 alleles, "++" indicates
the peptide binds to 10-19 alleles, and "+++" indicates the peptide
binds to 20-50 alleles. The 20% binding threshold is relative to a
theoretical maximum binding score, as calculated by an algorithm as
described by Stumiolo et al.
TABLE-US-00001 Selected peptides of B4 V regions predicted to bind
humans HLA-DR alleles. VH T cell VK T cell epitopes bind epitopes
bind (start pos.) freq. (start pos.) freq. (2) VQLQQPGAE + (2)
IVLTQSPAI +++ (12) VKPGASVRL + (3) VLTQSPAIM ++ (18) VRLSCKTSG +++
(19) VTMTCSASS + (36) WVKQRPGQG + (29) VNYMHWYQQ + (60) YNQKFKGRA +
(46) WIYDTSKLA ++ (64) FKGKAKLTV +++ (47) IYDTSKLAS + (80)
YMEVSSLTS ++ (93) VYYCARGSN +
These potentially immunogenic sequences in the B4 VH and B4 VK
polypeptides can be rendered less immunogenic by introducing
specific mutations that reduce or eliminate the binding of a
particular peptide to a human HLA-DR allele (see, for example
WO98/52976 and WO00/34317). Alternatively, non-human T-cell
epitopes are mutated so that they correspond to human self epitopes
that are present in human germline antibodies (see for example U.S.
Pat. No. 5,712,120).
Guidance for selecting appropriate mutations may be obtained by
reference to tertiary and quaternary structure of antibody variable
regions. Crystal structures of antibody variable domains are known
in the art and it is found that structures of the FRs are generally
very similar to one another. A theoretical model of the antibody
variable region of anti-CD19 antibody B4 VH0/VK0 can be constructed
from the most closely related antibody heavy and light chain
variable regions for which a structure has been determined, which
can be identified by from a primary structure alignment (Altschul
et al., (1990) J. Mol. Biol. 215:403-415). A threading algorithm is
used to model the B4 light and heavy chains onto the solved
structures (Marti-Renom et al., (2000) Annu Rev Biophys Biomol
Struct 29:291-325), and the threaded structures may be further
refined to obtain stereochemically favorable energies (Weiner et
al., (1984) J Am Chem Soc 106:765-784). It was found that the
solved heavy chain and light chain structures designated
respectively by their PDB database accession codes 1FBI (Fab
fragment of monoclonal antibody F9.13.7) and 1MIM (Fab fragment of
anti-CD25 chimeric antibody Sdz Chi621), were useful reference
structures for this purpose.
Preferred mutations do not unduly interfere with protein
expression, folding, or activity. According to the invention, amino
acids at positions Q5, V12, R19, L20, K23, T24, K38, R40, G42, Q43,
K65, K69, S85, S88, or V93 in B4 VH and amino acids at positions
Q1, V3, S7, I10, M11, V19, V29, S51, L53, A54, or S75 in B4 VK can
be mutated while still retaining the ability of the antibody to be
expressed and to bind to CD19 at levels comparable to the
unmodified form of B4. Thus the invention encompasses B4 antibodies
having at least one modification in the VH sequence selected from
the group of amino acid positions consisting of Q5, V12, R19, L20,
K23, T24, K38, R40, G42, Q43, K65, K69, S85, S88, and V93 and/or
having at least one modification in the VK sequence selected from
the group of amino acid positions consisting of Q1, V3, S7, I10,
M11, V19, V29, S51, L53, A54, and S75.
A nonexhaustive list of specific positions found to tolerate amino
acid substitutions according to the invention is presented in Table
2, together with exemplary substitutions at those positions.
TABLE-US-00002 TABLE 2 Substitutions in B4 V regions. Position in
B4 VH Substitution Position in B4 VK Substitution Gln5 Glu Gln1 Asp
Val12 Lys Val3 Ala Arg 19 Lys Ser7 Glu Leu20 Val Ile10 Thr Lys23
Glu, Asp Met11 Leu Thr24 Ala Val19 Ala Lys38 Arg Val 29 Ala Arg40
Ala, Thr Ser51 Asp Gly42 Asp, Glu Leu53 Thr Gln43 Lys Ala54 Asp
Lys65 Asp, Glu Ser75 Glu Lys69 Glu, Asp Ser85 Asp, Glu Ser88 Ala
Val93 Thr
One set of embodiments includes amino acid substitutions in the B4
VH polypeptide, selected from Q5E, V12K, R19K, L20V, K23E, T24A,
K38R, R40T, G42D, Q43K, K65D, K69B, S85D, S88A, and V93T.
Additionally contemplated substitutions are K23D, G42E, K65E and
K69D. Particular combinations of mutations are also found to be
useful. For example, in one specific embodiment, the substitutions
K23E and K69E are included. In another specific embodiment,
additionally the substitutions G42D and S88A are included, as
shown, for example, for B4 VHv1 (SEQ ID NO: 14). In yet another
specific embodiment, VHv1 additionally includes the substitutions
V12K and K65D (B4 VHv11) (SEQ ID NO:20). In a further specific
embodiment the substitutions Q5E, V12K, R19K, L20V, S85D, and S88A
am included, as exemplified by B4 VHv3 (SEQ ID NO:16). In yet a
further specific embodiment the substitutions Q5E, R19K, L20V,
R40T, Q43K, K65D, S85D, S88A, and V93T are included, as exemplified
by B4 VHv4 (SEQ ID NO:17). In yet a further specific embodiment the
substitutions Q5E, R19K, L20V, K38R, R40A, Q43K, K65D, S85D, and
V93T are included, as exemplified by B4 VHv5 (SEQ ID NO:18). In
another specific embodiment, substitutions of B4 VHv3 and B4 VHv4
are combined, as shown in the sequence of B4 VHv34 (SEQ ID
NO:21).
Another set of embodiments includes amino acid substitutions in the
B4 VK polypeptide, selected from Q1D, V3A, S7E, I10T, M11L, V19A,
V29A, S51D, L53T, A54D, and S75E. In one specific embodiment, the
substitution A54D is included. Particular combinations of mutations
are also found to be useful. For example, in more specific
embodiments, additionally the substitutions V3A and S7E are
included, as exemplified by B4 VKv1 (SEQ ID NO:26), or the
substitutions Q1D, I10T, and M11L are included, as exemplified, by
B4 VKv2 (SEQ ID NO:27). In a further embodiment, B4 VK1
additionally includes the substitutions V19A and S75E, as
exemplified in B4 VKv11 (SEQ ID NO:30). In yet another embodiment,
the substitutions I10T, M11L, V19A, V29A, and S75E are included, as
exemplified by B4 VKv3 (SEQ ID NO:28). In yet a further specific
embodiment, the substitutions I10T, M11L, V19A, S51D, and L53T are
included, as exemplified by B4 VKv4 (SEQ ID NO:29). In another
specific embodiment, substitutions of B4 VKv3 and B4 VKv4 are
combined, as shown in the sequence of B4 VKv34 (SEQ ID NO:31).
A primary structure alignment of some exemplary sequences of B4
VHvx and B4 VKvx of the invention, described above, are presented
in FIGS. 36 and 37, respectively. Amino acids depicted in bold are
positions of VH0 and VK0 that may be mutated according to the
invention, and underlined amino acids represent CDRs. VHv1-VHv34
and VKv1-VKv34 are representative heavy and light chains,
respectively, with specific amino acid substitutions that reduce
immunogenicity.
Variable region compositions of the invention include at least a
heavy chain or a light chain of the invention. For example, in one
embodiment, the variable region contains B4 VHv1 (SEQ ID NO:14) and
B4 VK0 (SEQ ID NO:25). In another embodiment, the variable region
contains B4 VHv1 (SEQ ID NO:14) and B4 VKv1 (SEQ ID NO:26). In yet
another embodiment, the variable region contains B4 VHv4 (SEQ ID
NO:17) and B4 VKv4 (SEQ ID NO:29). In yet another embodiment, the
variable region contains B4 VHv5 (SEQ ID NO:23) and B4 VKv4 (SEQ ID
NO:29). It is appreciated that other embodiments of the invention
are easily obtained by combinatorially matching the complete set of
B4 VHvx and B4 VKvy polypeptides contemplated by the invention.
Useful combinations are farther determined experimentally, by
analyzing protein compositions containing these combinations, such
as a B4 VHvx/VKvy antibody, for their expressability and CD-19
binding activity, as well as their reduced immunogenicity, as
described in more detail below.
Verification of the Reduced Immunogenicity of Variable Regions of
the Invention.
To verify that a mutation of the invention has indeed resulted in
reduced immunogenicity, standard experimental tests, which are well
known in the art, can be employed. For example, a T-cell
stimulation assay may be used (e.g. Jones et al. (2004), J.
Interferon Cytokine Res., 24:560). In such an assay, human
peripheral blood mononuclear cells (PBMCs) are obtained and
cultured according to standard conditions. After an optional
pre-stimulation, a peptide corresponding to a potential MHC Class
II epitope is added to the culture of PBMCs; the PBMCs are further
incubated, and at a later time tritiated thymidine is added. The
peptide can be a minimal 9-mer, or can have about 10 to 15, or more
than 15, amino acids. After further incubation of the cells,
incorporation of tritiated thymidine into DNA is then measured by
standard techniques.
The T-cell stimulation assay is thought to work by the following
mechanisms. First, if a peptide is used as a stimulator, the
peptide must first bind to an MHC Class II molecule present on a
cell among the PBMCs. Second, the MHC Class II/peptide complex must
interact productively with a T-cell receptor on a CD4+ T-cell. If
the test peptide is unable to bind sufficiently tightly to an MHC
Class II molecule, no signal will result. If the peptide is able to
bind an MHC Class II molecule and there are T-cells expressing an
appropriately rearranged T-cell receptor capable of recognizing a
particular MHC Class II/peptide complex, a signal should result.
However, if such T-cells have been deleted as a result of a
negative selection process, no signal will result. These mechanisms
are considered relevant to the immunogenicity of a protein
sequence, as inferred from the stimulation or lack of stimulation
by a given peptide.
If recognizing T-cells are present in very low numbers in the PBMC
population for stochastic reasons relating to failure of an
appropriate T-cell receptor to take place or proliferation of
other, unrelated T-cells followed by homeostasis of the T-cell
population, there may also be no signal even though a signal is
expected. Thus, false negative results may occur. Based on these
considerations, it is important to use a large number of different
sources of PBMCs and to test these samples independently. It is
also generally useful to test PBMCs from an ethnically diverse set
of humans, and to determine the MHC Class II alleles present in
each PBMC population.
The standard T-cell assay has the disadvantage that the tritium
incorporation signal is often only two-fold greater than the
background incorporation. The proteins and peptides of the
invention may also be tested in a modified T-cell assay in which,
for example, purified CD4+ T-cells and purified dendritic cells are
co-cultured in the presence of the test peptide, followed by
exposure to tritiated thymidine and then assayed for tritiated
thymidine incorporation. This second assay has the advantage that
tritiated thymidine incorporation into irrelevant cells, such as
CD8+ T-cells, is essentially eliminated and background is thus
reduced.
A third assay involves the testing of a candidate protein with
reduced immunogenicity in an animal such as a primate. Such an
assay would generally involve the testing of a B4 VHvx/VKvy protein
composition, such as an antibody, that had been designed by testing
individual component peptides for potential immunogenicity in a
cell-based assay such as one described above. Once such a candidate
B4 VHvx/VKvy protein composition is designed and expressed, the
protein is tested for immunogenicity by injection into an
animal.
Injection of the B4 VHvx/VKvy protein composition is generally
performed in the same manner as the anticipated route of delivery
during therapeutic use in humans. For example, intradermal,
subcutaneous, intramuscular, intraperitoneal injection or
intravenous infusion may be used. If more than one administration
is used, the administrations may be by different routes.
For immunogenicity testing purposes, it may be useful to
coadminister an adjuvant to increase the signal and minimize the
number of animals that need to be used. If an adjuvant is used, it
is possible to use an adjuvant lacking a protein component, such as
DNA with unmethylated CpG dinucleotides, bacterial lipid A,
N-formyl methionine, or other bacterial non-protein components.
Without wishing to be bound by theory, the rationale for avoiding
protein-containing adjuvants is that other proteins may provide
T-cell epitopes that will ultimately contribute to an antibody
response against the candidate protein.
After one or more administrations of the candidate B4 VHvx/VKvy
protein composition, the presence of anti-idiotype antibodies is
tested according to standard techniques, such as the ELISA method.
It is found that the B4 VHvx/VKvy protein compositions of the
invention induce antibody formation less frequently, and to a
lesser extent, than corresponding molecules containing original B4
VH/VK sequences.
Other Configurations of the Variable Regions of the Invention.
In addition to the use of the V regions of the invention in a naked
antibody, it is also possible to configure the V regions of the
invention into antibody fusion proteins that target toxins, immune
stimulators, and other proteins, as well as in Fabs, single-chain
Fvs, bispecific antibodies, and other configurations known in the
art of antibody engineering.
In certain embodiments of the invention, the light chain variable
region and the heavy chain variable region can be coupled,
respectively, to a light chain constant region and a heavy chain
constant region of an immunoglobulin. The immunoglobulin light
chains have constant regions that are designated as either kappa or
lambda chains. In a particular embodiment of the invention, the
light chain constant region is a kappa chain. The heavy chain
constant regions, and various modification and combinations thereof
are discussed below in more detail.
Fc Portion
The antibody variable domains of the present invention are
optionally fused to an Fc portion. As used herein, the Fc portion
encompasses domains derived from the heavy chain constant region of
an immunoglobulin, preferably a human immunoglobulin, including a
fragment, analog, variant, mutant or derivative of the constant
region. The constant region of an immunoglobulin heavy chain is
defined as a naturally-occurring or synthetically produced
polypeptide homologous to at least a portion of the C-terminal
region of the heavy chain, including the CH1, hinge, CH2, CH3, and,
for some heavy chain classes, CH4 domains. The "hinge" region joins
the CH1 domain to the CH2-CH3 region of an Fc portion. The constant
region of the heavy chains of all mammalian immunoglobulins exhibit
extensive amino acid sequence similarity. DNA sequences for these
immunoglobulin regions are well known in the art. (See, e.g.,
Gillies et al. (1989) J. Immunol. Meth. 125:191).
In the present invention, the Fc portion typically includes at
least a CH2 domain. For example, the Fc portion can include the
entire immunoglobulin heavy chain constant region
(CH1-hinge-CH2-CH3). Alternatively, the Fc portion can include all
or a portion of the hinge region, the CH2 domain and the CH3
domain.
The constant region of an immunoglobulin is responsible for many
important antibody effector functions, including those mediated by
Fc receptor (FcR) binding and by complement binding. There are five
major classes of the heavy chain constant region, classified as
IgA, IgG, IgD, IgE, and IgM, each with characteristic effector
functions designated by isotype.
IgG, for example, is separated into four .gamma. isotypes:
.gamma.1, .gamma.2, .gamma.3, and .gamma.4, also known as IgG1,
IgG2, IgG3, and IgG4, respectively. IgG molecules can interact with
multiple classes of cellular receptors including three classes of
Fc.gamma. receptors (Fc.gamma.R) specific for the IgG class of
antibody, namely Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII. The
sequences important for the binding of IgG to the Fc.gamma.R
receptors have been reported to be in the CH2 and CH3 domains.
Widely recognized effector functions of antibodies, particularly of
the IgG class, include complement-dependent cytotoxicity (CDC) and
antibody-dependent cellular cytotoxicity (ADCC). All of the IgG
subclasses (IgG1, IgG2, IgG3, IgG4) mediate CDC and ADCC to some
extent, with IgG1 and IgG3 being most potent for both activities
(Chapter 3, Table 3 in Paul, Essential Immunology 4.sup.th Ed., p.
62). CDC is believed to occur by multiple mechanisms; one mechanism
is initiated when an antibody binds to an antigen on a cell's
surface. Once the antigen-antibody complex is formed, the C1q
molecule is believed to bind the antigen-antibody complex. C1q then
cleaves itself to initiate a cascade of enzymatic activation and
cleavage of other complement proteins which then bind the target
cell surface and facilitate its depth through, for example, cell
lysis and/or ingestion by a macrophage. ADCC is believed to occur
when Fc receptors on cytotoxic cells, such as natural killer (NK)
cells, macrophages and neutrophils, bind to the Fc region of
antibodies bound to antigen on a cell's surface. Fc receptor
binding signals the cytotoxic cell to kill the target cell.
Characteristically, NK cells, believed to be the primary mediators
of ADCC, express only Fc.gamma.RIIIa.
It is often useful to alter the effector functions of an antibody.
For example, to treat cancers associated with a B cell malignancy
or to treat autoimmune diseases with a B cell component, it is
useful to enhance the ADCC activity of the antibody. It may be
particularly useful to enhance the ADCC activity of an antibody
directed to B cell surface antigens present at relatively low
density (Niwa et al., (2005) Clin. Cancer Res. 11:2327-2336), such
as to CD19. It is believed that the antigen density of CD19
relative to CD20 on the surface of B cells is roughly ten-fold
lower. Alterations in antibodies that increase the ADCC activity of
an antibody relative to its parent antibody are known in the art,
and generally correlate with modifications that increase the
binding affinity of the variant antibody to Fc.gamma.RIII (see for
example, U.S. Pat. No. 6,737,056). For example, mutations are
introduced into the Fc region at one or more positions (with
reference to their position in IgG.gamma.1) selected from 256, 290,
298, 312, 326, 330, 333, 334, 339, 360, 378 and 430 (numbering
according to Kabat et al. Sequences of Proteins of Immunological
Interest, 1991). Preferred mutations are at one or more positions
selected from 298, 333, and 334. For example, alanine substitutions
may be introduced.
ADCC activity of the antibody is also influenced by the particular
cell line used to produce the antibody. For example, antibodies
produced in the mouse myeloma NS/0 cells (or SP2/0 cells) generally
have low ADCC, and antibodies produced in rat myeloma YD cells (or
YB2/0) cells have high ADCC (Lifely et al., (1995) Glycobiology
5:813-822). It is known in the art that the type of cell line used
for antibody expression affects the carbohydrate structure of the
N-linked glycosyl chain, which is attached to the Fc region of the
antibody at position corresponding to N297 in IgG.gamma.1. The
carbohydrate structure of antibodies produced in CHO cells is
fucosylated, whereas the carbohydrate chain of antibodies produced
in YB2/0 is largely absent of fucose (Shinkawa et al., (2003) JBC
278:3466-3473). Antibodies that lack fucose on the carbohydrate
structure bind to human Fc.gamma.RIIIa with higher affinity
(Shields et al., (2002) JBC 277:26733-26740). In certain
embodiments, anti-CD19 antibodies with variable regions of the
invention are characterized by having reduced fucosylation on the
N-linked glycosyl chain of the Fc portion of the antibody.
It is also often useful to alter the serum half-life of the
antibody. The serum half-life of an antibody, as of an
immunoglobulin fusion protein, is influenced by the ability of that
antibody to bind to an Fc receptor (FcR) (Gillies et al., Cancer
Research (1999) 59:2159-66). The CH2 and CH3 domains of IgG2 and
IgG4 have undetectable or reduced binding affinity to Fc receptors
compared, to those of IgG1. Accordingly, the serum half-life of the
featured antibody can be increased by using the CH2 and/or CH3
domain from IgG2 or IgG4 isotypes. Alternatively, the antibody can
include a CH2 and/or CH3 domain from IgG1 or IgG3 with modification
in one or more amino acids in these domains to reduce the binding
affinity for Fc receptors (see, e.g., U.S. patent application Ser.
No. 09/256,156, published as U.S. patent application publication
2003-0105294).
The hinge region of the Fc portion normally adjoins the C-terminus
of the CH1 domain of the heavy chain constant region. When included
in the proteins of the present invention, the hinge is homologous
to a naturally-occurring immunoglobulin region and typically
includes cysteine residues linking two heavy chains via disulfide
bonds as in natural immunoglobulins. Representative sequences of
hinge regions for human and mouse immunoglobulin can be found in
ANTIBODY ENGINEERING, a PRACTICAL GUIDE, (Borrebaeck, ed., W. H.
Freeman and Co., 1992).
Suitable hinge regions for the present invention can be derived
front IgG1, IgG2, IgG3, IgG4, and other immunoglobulin isotypes.
The IgG1 isotype has two disulfide bonds in the hinge region
permitting efficient and consistent disulfide bonding formation.
Therefore, a preferred hinge region of the present invention is
derived from IgG1. Optionally, the first, most N-terminal cysteine
of an IgG1 hinge is mutated to enhance the expression and assembly
of antibodies or antibody fusion proteins of the invention (see,
e.g., U.S. patent application Ser. No. 10/093,958, published as
U.S. patent application publication 2003-0044423).
In contrast to IgG1, the hinge region of IgG4 is known to form
interchain disulfide bonds inefficiently (Angal et al., (1993),
Mol. Immunol. 30:105-8). Also, the IgG2 hinge region has four
disulfide bonds that tend to promote oligomerization and possibly
incorrect disulfide bonding during secretion in recombinant
systems. One suitable hinge region for the present invention can be
derived from the IgG4 hinge region, preferentially containing a
mutation that enhances correct formation of disulfide bonds between
heavy chain-derived moieties (Angal et al., (1993), Mol. Immunol.
30(1):105-8). Another preferred hinge region is derived from an
IgG2 hinge in which the first two cysteines are each mutated to
another amino acid, such as, in order of general preference,
serine, alanine, threonine, proline, glutamic acid, glutamine,
lysine, histidine, arginine, asparagine, aspartic acid, glycine,
methionine, valine, isoleucine, leucine, tyrosine, phenylalanine,
tryptophan or selenocysteine (see, e.g., U.S. patent application
publication 2003-0044423).
An Fc portion fused to an antibody variable region of the invention
can contain CH2 and/or CH3 domains and a hinge region that are
derived from different antibody isotypes. For example, the Fc
portion can contain CH2 and/or CH3 domains of IgG2 or IgG4 and a
hinge region of IgG1. Assembly of such hybrid Fc portions has been
described in U.S. patent application publication 2003-0044423.
When fused to an antibody variable region of the invention, the Fc
portion may contain one or more amino acid modifications that
generally extend the serum half-life of an Fc fusion protein. Such
amino acid modifications include mutations substantially decreasing
or eliminating Fc receptor binding or complement fixing activity.
For example, one type of such mutation removes the glycosylation
site of the Fc portion of an immunoglobulin heavy chain. In IgG1,
the glycosylation site is Asn297 (see, for example, U.S. patent
application Ser. No. 10/310,719, published as U.S. patent
application publication 2003-0166163).
The antibody variable regions of the invention can be attached to a
diagnostic and/or a therapeutic agent. The agent can be fused to
the antibody to produce a fusion protein. Alternatively, the agent
can be chemically coupled to the antibody to produce an
immuno-conjugate. The agent can be, for example, a toxin,
radiolabel, imaging agent, immunostimulatory moiety or the
like.
The antibody variable region of the invention can be attached to a
cytokine. Preferred cytokines include interleukins such as
interleukein-2 (IL-2), IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13,
IL-14, IL-15, IL-16, IL-18, IL-21, and IL-23, hematopoietic factors
such as granulocyte-macrophage colony stimulating factor (GM-CSF),
granulocyte colony stimulating factor (G-CSF) and erythropoeitin,
tumor necrosis factors (TNF) such as TNF.alpha., lymphokines such
as lymphotoxin, regulators of metabolic processes such as leptin,
interferons such as interferon .alpha., interferon .beta., and
interferon .gamma., and chemokines. Preferably, the
antibody-cytokine fusion protein or immunoconjugate displays
cytokine biological activity. In one embodiment, the antibody
variable domain is fused to IL-2. Preferably, several amino acids
within the IL-2 moiety are mutated to reduce toxicity, as described
in U.S. patent application publication 2003-0166163.
Optionally, the protein complexes can further include a second
agent, such as a second cytokine. In one embodiment, a B4 VHvx/VKvy
antibody fusion protein includes IL-12 and IL-2. The construction
of protein complexes containing an immunoglobulin domain and two,
different cytokines is described in detail in U.S. Pat. No.
6,617,135.
Antibody Production
Antibodies of the invention, as well as other variable
region-containing proteins of the invention, are produced by
methods well known in the art of protein engineering. Nucleic acid
vectors capable of expressing a heavy chain and a light chain which
include sequences of the invention are introduced into the
appropriate cell and the recombinant protein product is expressed
and purified. For example, antibodies of the invention can be
produced in engineered mammalian cell lines such as NS/0 cells, CHO
cells, SF2/0 cells (SP2/0-Ag14; ATCC-CRL 1581), YB2/0 cells
(YB2/3HL.P2.G11.16Ag.20; ATCC CRL-1662), or other mammalian cells
well known in the art of antibody production. In one embodiment, B4
VHvx/VKvy antibodies are produced in NS/0 cells. In another
embodiment, B4 VHvx/VKvy antibodies are produced in YB2/0 cells.
Alternatively, yeast, plants, insect cells, or bacteria may be used
to produce proteins containing variable regions of the
invention.
Administration
The antibodies of the invention are preferably used to treat
patients with B cell disorders such as B cell lymphomas or
autoimmune disorders with a B cell component such as rheumatoid
arthritis, myasthenia gravis, multiple sclerosis, systemic lupus
erythematosus, and so on. For B cell lymphoma, depending on the
judgment of the physician, it may be useful to treat a patient that
has failed other therapies. For example, some patients who are
treated with Rituxan.TM. may initially respond, but
Rituxan.TM.-resistant cancer cells may arise. Such patients should
generally still respond to the antibodies of the invention.
In the case of antibodies directed against CD19, it is sometimes
useful to clear the normal B cells from the body, as these cells
are likely to titrate the antibody of the invention. Rituxan.TM.
may be used for this purpose, according to standard procedures.
Alternatively, the anti-CD19 antibodies of the invention may be
used to clear the normal B cells from the body, for example as
described in Example 5 and Example 9.
Infusion is the preferred method of administration. Other methods
of administration include injection routes such as subcutaneous,
intradermal, intramuscular, intraperitoneal, or intravenous (bolus)
delivery. Inhalation and oral delivery are also possible methods of
delivery.
For a 70 kilogram human, a typical dose is in the range of about 50
milligrams to 2 grams, with a preferred dose in the range of about
400-600 milligrams. Dosing may be repeated about once every three
to six weeks, for example, during which normal B cells and tumor
cells are monitored.
Fusion proteins of the present invention are useful in treating
human disease, such as cancer. When treating cancer, it is for
example useful to administer an antibody-IL-2 fusion protein
comprising the variable regions of the invention by infusion or
subcutaneous injection, using doses of 0.1 to 100
milligrams/meter.sup.2/patient. In a preferred embodiment, it is
particularly useful to administer an antibody-IL-2 fusion protein
comprising the variable regions of the invention by infusion or
subcutaneous injection, using doses of 1 to 10
milligrams/meter.sup.2/patient, and more preferably about 3 to 6
milligrams/meter.sup.2/patient.
Pharmaceutical compositions of the invention may be used in the
form of solid, semisolid, or liquid dosage forms, such as, for
example, pills, capsules, powders, liquids, suspensions, or the
like, preferably in unit dosage forms suitable for administration
of precise dosages. The compositions include a conventional
pharmaceutical carrier or excipient and, in addition, may include
other medicinal agents, pharmaceutical agents, carriers, adjuvants,
etc. Such excipients may include other proteins, such as, for
example, human serum albumin or plasma proteins. Actual methods of
preparing such dosage forms are known or will be apparent to those
skilled in the art. The composition or formulation to be
administered will, in any event, contain a quantity of the active
component(s) in an amount effective to achieve the desired effect
in the subject being treated.
Administration of the compositions hereof can be via any of the
accepted modes of administration for agents that exhibit such
activity. These methods local or systemic administration.
Intravenous injection in a pharmaceutically acceptable carrier is a
preferred method of administration. The amount of active compound
administered will, of course, be dependent on the subject being
treated, the severity of the affliction, the manner of
administration, and the judgment of the proscribing physician.
The invention is further illustrated through the following
non-limiting examples.
Example 1. Construction of Anti-CD19 Antibodies Containing Variable
Region Heavy and Light Chains of the Invention
Standard genetic engineering techniques were used to introduce
nucleic acid sequences encoding a heavy chain region and light
chain region of the invention into an appropriate mammalian
expression vector. Exemplary cloning strategies are described
below. The expression vector pdHL12 is a later-generation pdHL
expression vector engineered to contain unique restriction sites
for the insertion of nucleic acid cassettes encoding heavy and
light chain variable regions, pdHL12 is designed to accept nucleic
acids encoding the heavy chain variable region as a Nhe I/Hind III
fragment, and nucleic acids encoding the light chain variable
region as an Afl II/Bam HI fragment, and to co-express intact
antibody heavy and light chains (see, for example U.S. patent
application 2003/0157054).
Nucleic acid sequences of heavy chain variable regions of the
invention, flanked by sequences with endonuclease restriction
recognition sequences 5'-CTTAAGC-3' (upstream, containing the Nhe I
site) and 5'-CGTAAGTGGATCC-3' (downstream, containing the Hind III
site), were synthesized de novo and inserted into a pUC
vector-derived carrier plasmid (Blue Heron Biotechnology, Bothell,
Wash.). The nucleic acid was excised from the carrier plasmid as a
Nhe I/Hind III fragment and ligated to the appropriate vector
fragment of a likewise digested pdHL12 plasmid. Nucleic acid
sequences for B4 VH0 (SEQ ID NO:1), B4 VHv1 (SEQ ID NO:2), B4 VHv2
(SEQ ID NO:3), B4 VHv3 (SEQ ID NO:4), B4 VHv4 (SEQ ID NO:5), B4
VHv5 (SEQ ID NO:6), and B4 VHv6 (SEQ ID NO:7) are shown.
Similarly, nucleic acids of light chain variable regions of the
invention, flanked by sequences with endonuclease restriction
recognition sequences 5'-GCTAGCTCCAGC-3' (upstream, containing the
Afl II site) and 5'-GGTAAGCTT-3' (downstream, containing the Bam HI
site), were synthesized de novo and inserted into a pUC
vector-derived carrier plasmid (Blue Heron Biotechnology, Bothell,
Wash.). The nucleic acid was excised from the carrier plasmid as an
Afl II/Bam HI fragment and ligated to the appropriate vector
fragment of a likewise digested pdHL12 plasmid. Nucleic acid
sequences encoding B4 VK0 (SEQ ID NO:8), B4 VKv1 (SEQ ID NO:9), B4
VKv2 (SEQ ID NO:10), B4 VKv1 (SEQ ID NO:11), and B4 VKv4 (SEQ ID
NO:12) are shown.
By inserting the nucleic acid sequences encoding the different
heavy and light chain variable regions combinatorially into pdHL12,
a panel of plasmids encoding B4 antibodies of the invention, B4
VHvx/VKvy, were generated.
Example 2. Expression and Purification of Antibodies of the
Invention
The following general techniques are used in the subsequent
Examples.
1A. Cell Culture and Transfection
To express antibodies transiently from mammalian cells, plasmid DNA
is introduced into human embryonic kidney 293 cells, or baby
hamster kidney (BHK) cells, by co-precipitation of plasmid DNA with
calcium phosphate and cells are grown without selection for plasmid
maintenance [Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, N.Y.].
Stably transfected clones are obtained by one of several standard
methods, for example, by electroporation or by nucleofection.
Electroporation of DNA into mouse myeloma NS/0 cells is performed
as follows. NS/0 cells are grown in Dulbecco's modified Eagle's
medium (DMEM, Life Technologies) supplemented with 10% fetal bovine
serum, 2 mM glutamine, 1 mM sodium pyruvate and 1.times.
penicillin/streptomycin. About 5.times.10.sup.6 cells are washed
once with PBS and resuspended in 0.5 ml phosphate buffer solution
(PBS). Ten micrograms of linearized plasmid DNA is then incubated
with the cells in a Gene Falser Cuvette (0.4 cm electrode gap,
BioRad) for 10 minutes on ice. Electroporation is performed using a
Gene Pulsar (BioRad) with settings at 0.25 V and microF. Cells are
allowed to recover for 10 minutes on ice, after which they are
resuspended in growth medium and then plated onto two 96-well
plates. Stably transfected clones are selected by growth in the
presence of 100 nM methotrexate (MTX), which is introduced two days
post-transfection. The cells are fed every 3 days for two to three
more times, and MTX-resistant clones generally appeared in 2 to 3
weeks. Supernatants from clones are assayed by anti-human Fc ELISA
to identify high producers [Gillies et al. (1989) J. Immunol.
Methods 125:191], High producing clones are isolated and propagated
in growth medium containing 100 nM MTX.
Similarly, other cell lines may be used to obtain stably
transfected clones by essentially the same method, such as CHO
cells, BHK cells, SP2/0 cells, and YB2/0 cells. When YB2/0 cells
were used, stably transfected clones were generally selected by
growth in the presence of 50 nM MTX.
Stably transfected clones, for example from rat myeloma YB2/0
cells, were also obtained by nucleofection. About 2.times.10.sup.6
YB2/0 cells, grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with heat inactivated 10% fetal bovine serum, 2 mM
glutamine, 1 mM sodium pyruvate, and 1.times.
penicillin/streptomycin, were centrifuged at 90.times.g at room
temperature for 10 min and resuspended in 100 .mu.l of supplemented
Nucleofector Solution V. 100 .mu.l of the cell suspension was mixed
with 2 .mu.g of linearized plasmid DMA (linearized, at the Fsp I
site in the .beta.-lactamase sequence), transferred into a cuvette
(Amaxa), and the nucleofection was performed using the appropriate
Nucleofector (Amaxa) program, Q-20, 500 .mu.l pre-warmed culture
medium was added and the sample was transferred into a well of a
12-well plate. One day post transfection, the cells were
resuspended in growth medium and plated onto 96 well plates at cell
densities ranging from approximately 10 cells/well to approximately
600 cells/well. Stably transfected clones were selected by growth
in the presence of 50 nM methotrexate (MTX), which was introduced
two days post-transfection. The cells were fed every 2 or 3 days
twice more, and MTX resistant clones generally appeared in 2 to 3
weeks. Supernatants from clones were assayed by anti-human Fc ELISA
to identify high producers [Gillies et al. (1989) J. Immunol.
Methods 125:191]. High producing clones were isolated and
propagated in growth medium containing 50 nM MTX.
The cells may be grown in an alternate medium known in the art,
such as HSFM supplemented with 2.5% fetal bovine serum and 100 nM
methotrexate, or a protein free medium such as CD medium.
1B. ELISAs
Different ELISAs are used to determine the concentrations of
protein products in the supernatants of MTX-resistant clones and
other test samples. For example, the anti-huFc ELISA is used to
measure the amount of human Fc-containing proteins, e.g., chimeric
antibodies, and the anti-hu kappa ELISA is used to measure the
amount of kappa light chain (of chimeric or human
immunoglobulins).
The anti-huFc ELISA is described in detail below.
A. Coating Plates
ELISA plates are coated with AffiniPure goat anti-human IgG (H+L)
(Jackson Immuno Research) at 5 microgram/ml in PBS, and 100
.mu.l/well in 90-well plates (Nunc-Immuno plate Maxisorp). Coated
plates are covered and incubated at 4.degree. C. overnight. Plates
are then washed 4 times with 0.05% Tween (Tween 20) in PBS, and
blocked with 1% BSA/1% goat serum in PBS, 200 microliter/well.
After incubation with the blocking buffer at 37.degree. C. for 2
hours, the plates are washed 4 times with 0.05% Tween in PBS and
tapped dry on paper towels.
B. Incubation with Test Samples and Secondary Antibody
Test samples are diluted to the proper concentrations in sample
buffer, which contains 1% BSA/1% goat serum/0.05% Tween in PBS. A
standard curve is prepared with a chimeric antibody (with a human
Fc), the concentration of which is known. To prepare a standard
curve, serial dilutions are made in the sample buffer to give a
standard curve ranging from 125 ng/ml to 3.9 ng/ml. The diluted
samples and standards are added to the plate, 100 microliter/well,
and the plate is incubated at 37.degree. C. for 2 hours.
After incubation, the plate is washed 8 times with 0.05% Tween in
PBS. To each well is then added 100 microliter of the secondary
antibody, the horseradish peroxidase (HRP)-conjugated anti-human
IgG (Jackson Immuno Research), diluted around 1:120,000 in the
sample buffer. The exact dilution of the secondary antibody has to
be determined for each lot of the HRP-conjugated anti-human IgG.
After incubation at 37.degree. C. for 2 hours, the plate is washed
8 times with 0.03% Tween in PBS.
C. Development
The substrate solution is added to the plate at 100 .mu.l/well. The
substrate solution is prepared by dissolving 30 mg of
o-phenylenediamine dihydrochloride (OPD) (1 tablet) into 15 ml of
0.025 M citric acid/0.05M Na.sub.2HPO.sub.4 buffer, pH 5, which
contains 0.03% of freshly added H.sub.2O.sub.2. The color is
allowed to develop for 30 minutes at room temperature in the dark.
The developing time is subject to change, depending on lot to lot
variability of the coated plates, the secondary antibody, etc. The
color development in the standard curve is observed to determine
when to stop the reaction. The reaction is stopped by adding 4N
H.sub.2SO.sub.4, 100 .mu.l/well. The plate is read by a plate
reader, which is set at both 490 nm and 650 nm and programmed to
subtract off the background OD at 650 nm from the OD at 490 nm.
The anti-hu kappa ELISA follows the same procedure as described
above, except that the secondary antibody used is horseradish
peroxidase-conjugated goat anti-hu kappa (Southern Biotechnology
Assoc. Inc., Birmingham, Ala.), used at a 1:4000 dilution.
Purification
Standard antibody purification procedures were followed. Typically,
B4 VHvx/VKvy antibody compositions of the invention were purified
from cell-culture supernatants via Protein A chromatography based
on the affinity of the Fc portion for Protein A. The conditioned
supernatant from cells expressing B4 VHvx/VKvy antibody
compositions was loaded onto a pre-equilibrated Fast-Flow Protein A
Sepharose column. The column was washed extensively with sodium
phosphate buffer (50 mM Sodium Phosphate, 150 mM NaCl at neutral
pH). Bound protein was eluted by a low pH (pH 2.5-3) sodium
phosphate buffer (composition as above) and the eluted fractions
were immediately neutralized to about pH 6.5 with 1M Tris base. The
compositions were stored in 50 mM Sodium Phosphate, 150 mM NaCl, pH
6.5 supplemented with Tween 80 to 0.01%.
The purity and integrity of the product was routinely assessed by
HPLC size exclusion chromatography and by SDS-PAGE. Results showed
that the B4 VHvx/VKvy antibodies of the invention were typically
greater than 90% non-aggregated and intact, with little evidence of
degradation products.
Example 3. Determination of the Relative Binding Affinity of B4
Antibodies of the Invention to CD-19 Presenting Cells
To ascertain that the antibodies of the invention retained binding
to CD19 a competition assay was used, in which the strength of
these antibodies to inhibit binding of labeled parental B4 antibody
(B4-VH0/VK0) to Daudi lymphoma cells, which bear the CD19 antigen,
was measured.
Biotin-labeled B4 VH0/VK0 antibody was prepared using the EZ-link
Sulfo-NHS-LC-Biotinylation Kit (Pierce, #21430) according to the
supplied protocol. The product was dialyzed with a Slide-a-lyzer
(Pierce, #66425), and analyzed by HPLC size exclusion
chromatography.
Briefly, a titration series was prepared of biotin-labeled B4
VH0/VK0 antibody pre-mixed at a final concentration of 100 ng/ml
with one of the unlabeled, experimental B4 VHvx/VKvy antibodies at
800 ng/ml, 400 ng/ml, 200 ng/ml, 100 ng/ml, and 50 ng/ml in a
PBS/2% serum buffer. As a control, the biotin-labeled antibody was
pre-mixed with unlabeled B4 VH0/VK0 antibody at the same
concentrations as above (inhibition control) or with buffer only
(positive binding control). The combined antibodies were added to
Daudi cells for 30 minutes at 4.degree. C. A 1:200 dilution of
FITC-labeled streptavidin was added to the cells and the samples
were incubated for a further 30 minutes at 4.degree. C. Bound
amount of labeled B4 VH0/VK0 antibody was quantitated by FACS
analysis, and the results were expressed, as "percent inhibition,"
relative to the positive binding control. The tested antibodies
were B4 VH0/VK0 (C), B4 VHv1/VKv1 (1), B4 VHv2/VKv1 (2), B4
VHv1/VKv2 (3), B4 VHv2/VKv2 (4), B4 VHv3/VKv3 (5), B4 VHv4/VKv3
(6), B4 VHv3/VKv4 (7), B4 VHv4/VKv4 (8), B4 VHv5/VKv4 (9), and B4
VHv6/VKv4 (10). Representative results of two experiments are shown
in Table 3 and Table 4.
TABLE-US-00003 TABLE 3 Inhibition of Biotin-B4 VH0/VK0 binding to
Daudi cells by antibodies of the invention. Antibody Ratio (%
inhibition of labeled B4 binding) (unlabeledl/labeled) C 1 2 3 4 5
6 7 8 8x 68 56 56 56 56 60 56 60 60 4x 56 44 40 44 44 40 44 52 48
2x 44 28 28 32 32 36 32 36 32 1x 28 16 16 20 16 20 16 20 16 0.5x 16
12 8 12 8 12 12 12 12
TABLE-US-00004 TABLE 4 Inhibition of Biotin-B4 VH0/VK0 binding to
Daudi cells by antibodies of the invention Antibody Ratio (%
inhibition of labeled 34 binding) (unlabeled/labeled) C 9 10 8x 93
90 87 4x 90 87 80 2x 83 70 70 1x 67 57 60 0.5x 50 47 53
As shown in Table 3 and Table 4, it was found that the B4 VHvx/VKvy
antibodies inhibited binding of labeled B4 antibody to Daudi cells
to a similar extent as the unlabeled B4 VH0/VK0 antibody did,
indicating that the affinities of the B4 VHvx/VKvy antibodies and
B4 VH0/VK0 antibody are similar.
Example 4. ADCC Activity of Antibodies of the Invention
ADCC activity mediated by the antibodies of the invention produced
in various cell lines was assessed. ADCC was determined by a
standard .sup.51Cr release assay, as practiced in the art. A serial
dilution of the antibodies was prepared (4-fold dilutions in a
range from 100 ng/ml to 0.025 ng/ml), and lysis by purified human
PBMCs (effector cells) of .sup.51Cr-labeled Daudi cells (target;
E:T is 100:1) in the presence of the antibodies was measured by
specific .sup.51Cr release, relative to total cellular .sup.51Cr
(adjusting for spontaneously released .sup.51Cr). B4 VHv4/VKv4
antibodies produced from human embryonic kidney 293T cells or from
YB2/0 cells, and B4 VHv5/VKv4 antibodies produced from a NS/0 cell
line or from YB2/0 cells were tested.
FIG. 38 shows the result of such an experiment. Both antibodies
obtained from YB2/0 cell expression were equally active in
mediating ADCC, and at least 50 fold more active than the
corresponding antibody obtained by expression from a NS/0 cell line
or from 293T cells. B4 VHv4/VKv4 produced from 293T cells was more
active than B4 VHv5/VKv4 produced from a NS/0 cell line.
Example 5. Depletion of Human B Cells Grafted into SCID Mice by
Antibodies of the Invention
The depletion of B cells is useful in a number of therapeutic
contexts. For example, antibody-driven autoimmune and inflammatory
disorders may be treated with antibodies of the invention to reduce
or essentially eliminate B cells. Alternatively, when treating with
a tumor-targeting agent such as Zevalin.TM. or Bexxar.TM. or a
Leu16-IL2 fusion protein (WO2005/016969), it is useful to first
eliminate normal B cells.
To address whether an antibody of the invention could be used to
deplete human B cells, the following experiment was performed. On
day 0, male SCID CB17 mice (n=3) were engrafted with purified human
PBMCs in which about 4.5.times.10.sup.7 cells in 0.2 mls of PBS
were injected intraperitoneally, essentially following a protocol
described for the transfer of human spleen cells (Yacoub-Youssef et
al., Transpl. Immunol. (2005) 15:157-164). On day 3, the mice were
injected intraperitoneally with either PBS or 50 micrograms of the
anti-CD20 antibody Leu16 or with the K4H4 anti-CD19 antibody.
Levels of human IgM were measured by a human IgM ELISA quantitation
kit (Bethyl Laboratories; Cat #E80-100) on days 7, 14, and 21.
FIG. 39 shows typical results. In the PBS-treated controls, the
titer of human IgM steadily increased, reaching about 800
micrograms/ml on day 42. In the mice treated with either Leu16 or
B4 VHv5/VKv4 antibody, human IgM titers were essentially absent,
indicating that human B cells were depleted by these antibody
treatments.
Example 6. Treatment of a Lymphoma-Bearing Mammal With an Antibody
of the Invention
To address whether the antibodies of the invention were functional
in vivo, the B4 VHv4/VKv4 antibody, expressed in YB2/0 cells, was
tested in an animal model of human lymphoma. Eight-week-old SCID
CB17 mice (n=6) were injected intravenously with about
1.times.10.sup.6 viable `Namalwa` Nalm-6-UM-1 cells on day 0. On
days 1, 3 and 5, mice were treated with 500 micrograms of antibody
intraperitoneally or with PBS. About one to two times per week the
mice were examined to see which mice had become ill enough to
require euthanasia.
FIG. 40 shows typical results of such an experiment. Mice treated
with PBS all became ill within 10 weeks of the injection of the
tumor cells, while mice treated with the B4 VHv4/VKv4 antibody
became ill at later times, and three of the six mice in this group
remained healthy throughout the 30-week course of the
experiment.
Example 7. Treatment of a Burkitt's Lymphoma-Bearing Mammal With an
Antibody of the Invention in Combination With Chemotherapy
To address whether the antibodies of the invention could be used in
vivo in combination with chemotherapy, the B4 VHv4/VKv4 antibody
was tested in animal models of human lymphoma that were more
stringent than in the previous example, corresponding to more
advanced or harder to treat forms of lymphoma. In one
representative experiment, the Daudi cell line, which is a
Burkitt's lymphoma cell line, was treated with the B4 VHv4/VKv4
antibody, expressed in YB2/0 cells, with or without
cyclophosphamide (CPA). Eight-week-old SCID CB17 mice (n=6) were
injected intravenously with about 5.times.10.sup.6 viable Daudi
cells on day 0. On days 8 and 12, mice were treated with 100
micrograms of antibody or PBS. On day 7, the mice were treated with
PBS or 75 micrograms of CPA per kilogram of body weight. Treatments
were administered intraperitoneally in 0.2 mls. About one to two
times per week the mice were examined to see which mice had become
ill enough to require euthanasia.
FIG. 41 shows the results of a typical experiment. In the control
group treated with neither antibody nor CPA, all of the mice became
ill within four weeks of injection of the lymphoma cells. In the
groups of mice singly treated with either the B4 VHv4/VKv4 antibody
or CPA, 5 out of the 6 mice were healthy for at least five weeks
but became ill within about six weeks. In the group of mice treated
with both B4 VHv4/VKv4 antibody and CPA, all of the mice remained
healthy for at least eight weeks.
Example 8. Treatment of Lymphoma Disseminated Disease With an
Antibody of the Invention Combined With Chemotherapy
In another set of experiments, Namalwa cells were injected
intravenously into mice as described in the previous Example,
except that 2.times.10.sup.6 cells were used instead of
1.times.10.sup.6 cells. This increased number of cells results in a
more aggressive disease course, as indicated by a comparison of the
PBS-treated mice in FIG. 42, which all became ill within three
weeks, as opposed to the PBS-treated mice in FIG. 40, which became
ill between five and 10 weeks. Mice (n=5) were treated with 500
micrograms of antibody intraperitoneally or with PBS on days 3, 7
and 11. Mice were also treated with either cyclophosphamide (75
mg/kg, i.p.), or vincristine (0.4 mg/kg, i.v.) or doxorubicin (3
mg/kg, i.v.) or PBS (i.v.) on days 3, 7, and 11. The results are
shown in FIG. 42(a-c). The results indicated that each of the three
chemotherapeutic agents could be combined with an antibody of the
invention.
Example 9. Treatment of a Human Patient With Antibodies and Methods
of the Invention
The anti-CD19 antibodies of the invention are used to treat human
diseases and disorders as follows. In general, the preferred method
of administration is by intravenous infusion or intravenous
injection, although subcutaneous injection, inhalation, oral
delivery, and other methods are also possible. Administration about
once every 2, 3 or 4 weeks is used, although the frequency of
administration may vary depending on the needs of the patient. A
typical dose is about 100 to 800 mgs for an adult human. Treated
patients are monitored for signs of infection that may result from
immunosuppression.
For example, a patient with Castleman's disease is treated with the
anti-CD19 B4 VHv4/VKv4 antibody of the invention about once every
two weeks at a dose of about 8 mg/kg, with administration by drip
infusion for about 1 hour.
A patient with rheumatoid arthritis is treated with the anti-CD19
B4 VHv4/VKv4 antibody about once every four weeks at a dose of
about 8 mg/kg, with administration by drip infusion for about 1
hour. Progression of joint destruction is found to be significantly
inhibited by monotherapy, even when compared to disease-modifying
anti-rheumatic drugs.
A patient with Crohn's disease is treated with the anti-CD19 B4
VHv4/VKv4 antibody about once every four weeks at a dose of about 8
mg/kg, with administration by drip infusion for about 1 hour.
A patient with multiple myeloma is treated with the anti-CD19 B4
VHv4/VKv4 antibody about once every three weeks at a dose of about
8 mg/kg, with administration by drip infusion, for about 1 hour.
Treatment with the anti-CD19 B4 VHv4/VKv4 is combined with a
standard-of-care treatment for multiple-myeloma as determined by a
physician as appropriate for the patient.
A patient with a B cell lymphoma is treated with the anti-CD19 B4
VHv4/VKv4 antibody about once every three weeks at a dose of about
8 mg/kg, with administration by drip infusion for about 1 hour,
optionally in combination with an antibody such as Rituxan.TM. at
about 375 milligrams per square meter of body surface area, which
is administered every week, or with the anti-CD22 antibody
epratuzamab. Alternatively, in the case of a patient with
refractory lymphoma, treatment with the anti-CD19 B4 VHv4/VKv4
antibody is combined with a radioimmunoconjugate such as Bexxar.TM.
or Zevalin.TM..
More specifically, a patient with a B cell lymphoma is treated with
the anti-CD19 B4 VHv4/VKv4 antibody about once every three weeks at
a dose of about 8 mg/kg, with administration by drip infusion for
about 1 hour, optionally in combination with a chemotherapeutic
regimen such as cyclophosphamide plus vincristine plus doxorubicin
plus prednisolone ("CHOP"), or CHOP plus bleomycin, or CHOP plus
etoposide, or mitoxantrone plus vincristine plus thiotepa, or
etoposide plus prednisolone plus cytarabin plus cisplatin, or mesna
plus ifosfamide plus mitoxantrone plus etoposide, or bendamustin,
or fludaribin and 2-CdA.
In an alternative treatment strategy, a patient with a B cell
lymphoma is initially treated with the anti-CD19 B4 VHv4/VKv4
antibody at a dose of about 8 mg/kg, and is then later treated with
an anti-CD20-IL2 fusion protein such as that described in
WO2005/016969. For example, a patient is treated on day 1 with the
anti-CD19 B4 VHv4/VKv4 antibody with administration by drip
infusion for about 1 hour, and then treated on day 2 and day 4 with
an anti-CD20-IL2 fusion protein at a dose of about 150 micrograms
per kg with administration by drip infusion for about 4 hours, and
this cycle is repeated about every 3 weeks. Without wishing to be
bound by theory, the anti-CD19 B4 VHv4/VKv4 antibody has the effect
of clearing most of the normal B cells from the patient, so that
the anti-CD20-IL2 fusion protein exerts its effects by binding to
remaining tumor cells.
Equivalents
The indention may be embodied in other specific forms without
departing from the spirit or essential characteristics thereof. The
foregoing embodiments are therefore to be considered in all
respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes which come within the meaning and range of equivalency of
the claims are intended to be embraced therein.
SEQUENCE LISTINGS
1
491360DNAHomo sapiens 1caggtgcaac tgcagcagcc tggggctgaa gtggtgaagc
ctggggcttc agtgagactg 60tcctgcaaga cttctggcta caccttcacc agcaactgga
tgcactgggt gaagcagagg 120cctggacaag gccttgagtg gatcggagag
attgatcctt ctgatagtta tactaactac 180aatcaaaagt tcaagggcaa
ggccaagttg actgtagaca aatcctccag cacagcctac 240atggaagtca
gcagcctgac atctgaggac tctgcggtct attactgtgc aagaggtagc
300aacccttact actatgctat ggactactgg ggtcaaggaa cctcagtcac
cgtctcctca 3602360DNAArtificial SequenceNucleic acid sequence
encoding B4 antibody heavy chain variable region with codons for
amino acid substitutions K23E, G42D, K69E, and S85D (B4 VHv1)
2caggtgcaac tgcagcagcc tggggctgaa gtggtgaagc ctggggcttc agtgagactg
60tcctgcgaga cttctggcta caccttcacc agcaactgga tgcactgggt gaagcagagg
120cctgaccaag gacttgagtg gatcggagag attgatcctt ctgatagtta
tactaactac 180aatcaaaagt tcaagggcaa ggccgaattg actgtagaca
aatcctccag cacagcctac 240atggaagtca gcgacctgac atctgaggac
tctgcggtct attactgtgc aagaggtagc 300aacccttact actatgctat
ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 3603360DNAArtificial
SequenceNucleic acid sequence encoding B4 antibody heavy chain
variable region with codons for amino acid substitutions K69E, and
S85D (B4 VHv2) 3caggtgcaac tgcagcagcc tggggctgaa gtggtgaagc
ctggggcttc agtgagactg 60tcctgcaaga cttctggcta caccttcacc agcaactgga
tgcactgggt gaagcagaga 120cctggacaag gacttgagtg gatcggagag
attgatcctt ctgatagtta tactaactac 180aatcaaaagt tcaagggcaa
ggccgaattg actgtagaca aatcctccag cacagcctac 240atggaagtca
gcgacctgac atctgaggac tctgcggtct attactgtgc aagaggtagc
300aacccttact actatgctat ggactactgg ggtcaaggaa cctcagtcac
cgtctcctca 3604360DNAArtificial SequenceNucleic acid sequence
encoding B4 antibody heavy chain variable region with codons for
amino acid substitutions Q5E, V12K, R19K, L20V, T24A, S85D, and
S88A (B4 VHv3) 4caggtgcaac tggagcagcc tggggctgaa gtgaagaagc
ctggggcttc agtgaaggtg 60tcctgcaagg cttctggcta caccttcacc agcaactgga
tgcactgggt gaagcagagg 120cctggacaag gacttgagtg gatcggagag
attgatcctt ctgatagtta tactaactac 180aatcaaaagt tcaagggcaa
ggccaagttg actgtagaca aatcctccag cacagcctac 240atggaagtca
gcgacctgac agctgaggac tctgcggtct attactgtgc aagaggtagc
300aacccttact actatgctat ggactactgg ggtcaaggaa cctcagtcac
cgtctcctca 3605360DNAArtificial SequenceNucleic acid sequence
encoding B4 antibody heavy chain variable region with codons for
amino acid substitutions Q5E, R19K, L20V, R40T, Q43K, K65D, S85D,
S88A, and V93T (B4 VHv4) 5caggtgcaac tggagcagcc tggggctgaa
gtggtgaagc ctggggcttc agtgaaggtg 60tcctgcaaga cttctggcta caccttcacc
agcaactgga tgcactgggt gaagcagacg 120cctggaaaag gacttgagtg
gatcggagag attgatcctt ctgatagtta tactaactac 180aatcaaaagt
tcgatggcaa ggccaagttg actgtagaca aatcctccag cacagcctac
240atggaagtca gcgacctgac agctgaggac tctgcgacct attactgtgc
aagaggtagc 300aacccttact actatgctat ggactactgg ggtcaaggaa
cctcagtcac cgtctcctca 3606360DNAArtificial SequenceNucleic acid
sequence encoding B4 antibody heavy chain variable region with
codons for amino acid substitutions Q5E, V12K, R19K, L20V, T24A,
K38R, R40A, Q43K, K65D, S85D, and V93T (B4 VHv5) 6caggtgcaac
tggagcagcc tggggctgaa gtgaagaagc ctggggcttc agtgaaggtg 60tcctgcaagg
cttctggcta caccttcacc agcaactgga tgcactgggt gagacaggca
120cctggaaaag gacttgagtg gatcggagag attgatcctt ctgatagtta
tactaactac 180aatcaaaagt tcgatggcaa ggccaagttg actgtagaca
aatcctccag cacagcctac 240atggaagtca gcgacctgac atctgaggac
tctgcgacct attactgtgc aagaggtagc 300aacccttact actatgctat
ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 3607360DNAArtificial
SequenceNucleic acid sequence encoding B4 antibody heavy chain
variable region with codons for amino acid substitutions Q5E, R19K,
L20V, K65D, S85D, and V93T (B4 VHv6) 7caggtgcaac tggagcagcc
tggggctgaa gtggtgaagc ctggggcttc agtgaaggtg 60tcctgcaaga cttctggcta
caccttcacc agcaactgga tgcactgggt gaagcagagg 120cctggacaag
gacttgagtg gatcggagag attgatcctt ctgatagtta tactaactac
180aatcaaaagt tcgatggcaa ggccaagttg actgtagaca aatcctccag
cacagcctac 240atggaagtca gcgacctgac atctgaggac tctgcgacct
attactgtgc aagaggtagc 300aacccttact actatgctat ggactactgg
ggtcaaggaa cctcagtcac cgtctcctca 3608312DNAHomo sapiens 8caaattgttc
tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60atgacctgca
gtgccagctc aggtgtcaac tacatgcact ggtaccagca gaagccaggc
120acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt
ccctgctcgc 180ttcagtggca gtgggtctgg gacctcttat tctctcacaa
tcagcagcat ggaggctgaa 240gatgctgcca cttattactg ccatcagcga
ggtagttaca cgttcggagg ggggaccaag 300ctggaaataa aa
3129312DNAArtificial SequenceNucleic acid sequence encoding B4
antibody light chain variable region with codons for amino acid
substitutions V3A, S7E, and A54D (B4 VKv1) 9caaattgctc tcacccagga
gccagcaatc atgtctgcat ctccagggga gaaggtcacc 60atgacctgca gtgccagctc
aggtgtcaac tacatgcact ggtatcagca gaagccaggc 120acctccccca
aaagatggat ttatgacaca tccaaactgg attctggagt ccctgctcgc
180ttcagtggca gtgggtctgg gacctcttat tctctcacaa tcagcagcat
ggaggctgaa 240gatgctgcca cttattactg ccatcagcga ggtagttaca
cgttcggagg ggggaccaag 300ctggaaataa aa 31210312DNAArtificial
SequenceNucleic acid sequence encoding B4 antibody light chain
variable region with codons for amino acid substitutions Q1D, I10T,
M11L, and A54D (B4 VKv2) 10gacattgttc tcacccagtc tccagcaact
ttgtctgcat ctccagggga gaaggtcacc 60atgacctgta gtgccagctc aggtgtcaac
tacatgcact ggtatcagca gaagccaggc 120acctccccca aaagatggat
ttatgacaca tccaaactgg attctggagt ccctgctcgc 180ttcagtggca
gtgggtctgg gacctcttat tctctcacaa tcagcagcat ggaggctgaa
240gatgctgcca cttattactg ccatcagcga ggtagttaca cgttcggagg
ggggaccaag 300ctggaaataa aa 31211312DNAArtificial SequenceNucleic
acid sequence encoding B4 antibody light chain variable region with
codons for amino acid substitutions I10T, M11L, V19A, V29A, and
S75E (B4 VKv3) 11caaattgttc tcacccagtc tccagcaact ttgtctgcat
ctccagggga gaaggctacc 60atgacctgca gtgccagctc aggtgctaac tacatgcact
ggtaccagca gaagccaggc 120acctccccca aaagatggat ttatgacaca
tccaaactgg cttctggagt ccctgctcgc 180ttcagtggca gtgggtctgg
gacctcttat tctctcacaa tcgagagcat ggaggctgaa 240gatgctgcca
cttattactg ccatcagcga ggtagttaca cgttcggagg ggggaccaag
300ctggaaataa aa 31212312DNAArtificial SequenceNucleic acid
sequence encoding B4 antibody light chain variable region with
codons for amino acid substitutions I10T, M11L, V19A, S51D, and
L53T (B4 VKv4) 12caaattgttc tcacccagtc tccagcaact ttgtctgcat
ctccagggga gaaggctacc 60atgacctgta gtgccagctc aggtgtcaac tacatgcact
ggtaccagca gaagccaggc 120acctccccca aaagatggat ttatgacaca
gacaaaacgg cttctggagt ccctgctcgc 180ttcagtggca gtgggtctgg
gacctcttat tctctcacaa tcagcagcat ggaggctgaa 240gatgctgcca
cttattactg ccatcagcga ggtagttaca cgttcggagg ggggaccaag
300ctggaaataa aa 31213120PRTHomo sapiens 13Gln Val Gln Leu Gln Gln
Pro Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Leu
Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30 Trp Met
His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50
55 60 Lys Gly Lys Ala Lys Leu Thr Val Asp Lys Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Glu Val Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Asn Pro Tyr Tyr Tyr Ala Met
Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115
120 14120PRTArtificial SequenceAmino acid sequence of B4 antibody
heavy chain variable region with codons for amino acid
substitutions K23E, G42D, K69E, and S85D (B4 VHv1) 14Gln Val Gln
Leu Gln Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser
Val Arg Leu Ser Cys Glu Thr Ser Gly Tyr Thr Phe Thr Ser Asn 20 25
30 Trp Met His Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile
35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln
Lys Phe 50 55 60 Lys Gly Lys Ala Glu Leu Thr Val Asp Lys Ser Ser
Ser Thr Ala Tyr 65 70 75 80 Met Glu Val Ser Asp Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Asn Pro Tyr Tyr
Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val
Ser Ser 115 120 15120PRTArtificial SequenceAmino acid sequence of
B4 antibody heavy chain variable region with codons for amino acid
substitutions K69E, and S85D (B4 VHv2) 15Gln Val Gln Leu Gln Gln
Pro Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Leu
Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30 Trp Met
His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50
55 60 Lys Gly Lys Ala Glu Leu Thr Val Asp Lys Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Glu Val Ser Asp Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Asn Pro Tyr Tyr Tyr Ala Met
Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115
120 16120PRTArtificial SequenceAmino acid sequence of B4 antibody
heavy chain variable region with codons for amino acid
substitutions Q5E, V12K, R19K, L20V, T24A, S85D, and S88A (B4 VHv3)
16Gln Val Gln Leu Glu Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Asn 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn
Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Lys Leu Thr Val Asp
Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Val Ser Asp Leu Thr
Ala Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Asn
Pro Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser
Val Thr Val Ser Ser 115 120 17120PRTArtificial SequenceAmino acid
sequence of B4 antibody heavy chain variable region with codons for
amino acid substitutions Q5E, R19K, L20V, R40T, Q43K, K65D, S85D,
S88A, and V93T (B4 VHv4) 17Gln Val Gln Leu Glu Gln Pro Gly Ala Glu
Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr
Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30 Trp Met His Trp Val Lys
Gln Thr Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp
Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Asp Gly
Lys Ala Lys Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Glu Val Ser Asp Leu Thr Ala Glu Asp Ser Ala Thr Tyr Tyr Cys 85
90 95 Ala Arg Gly Ser Asn Pro Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly
Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120
18120PRTArtificial SequenceAmino acid sequence of B4 antibody heavy
chain variable region with codons for amino acid substitutions Q5E,
V12K, R19K, L20V, T24A, K38R, R40A, Q43K, K65D, S85D, and V93T (B4
VHv5) 18Gln Val Gln Leu Glu Gln Pro Gly Ala Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Asn 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr
Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Asp Gly Lys Ala Lys Leu Thr
Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Val Ser Asp
Leu Thr Ser Glu Asp Ser Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly
Ser Asn Pro Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly
Thr Ser Val Thr Val Ser Ser 115 120 19120PRTArtificial
SequenceAmino acid sequence of B4 antibody heavy chain variable
region with codons for amino acid substitutions Q5E, R19K, L20V,
K65D, S85D, and V93T (B4 VHv6) 19Gln Val Gln Leu Glu Gln Pro Gly
Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Thr Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30 Trp Met His Trp
Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu
Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60
Asp Gly Lys Ala Lys Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65
70 75 80 Met Glu Val Ser Asp Leu Thr Ser Glu Asp Ser Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Gly Ser Asn Pro Tyr Tyr Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120
20120PRTArtificial SequenceAmino acid sequence of B4 antibody heavy
chain variable region with codons for amino acid substitutions
V12K, K23E, G42D, K65D, K69E, and S85D (B4 VHv11) 20Gln Val Gln Leu
Gln Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val
Arg Leu Ser Cys Glu Thr Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile 35
40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys
Phe 50 55 60 Asp Gly Lys Ala Glu Leu Thr Val Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Met Glu Val Ser Asp Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Asn Pro Tyr Tyr Tyr
Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser
Ser 115 120 21120PRTArtificial SequenceAmino acid sequence of B4
antibody heavy chain variable region with codons for amino acid
substitutions Q5E, V12K, R19K, L20V, T24A, R40T, Q43K, K65D, S85D,
S88A, and V93T (B4 VHv34) 21Gln Val Gln Leu Glu Gln Pro Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30 Trp Met His Trp Val Lys
Gln Thr Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp
Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Asp Gly
Lys Ala Lys Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Glu Val Ser Asp Leu Thr Ala Glu Asp Ser Ala Thr Tyr Tyr Cys 85
90 95 Ala Arg Gly Ser Asn Pro Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly
Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120
2230PRTArtificial SequenceAmino acid sequence of B4 antibody heavy
chain framework region 1 with variable amino acid residues X5, X12,
X19, X20, X23, and X24 (B4 VHfr1)MISC_FEATURE(5)..(5)Xaa is Gln or
GluMISC_FEATURE(12)..(12)Xaa is Val or LysMISC_FEATURE(19)..(19)Xaa
is Arg or LysMISC_FEATURE(20)..(20)Xaa is Leu or
ValMISC_FEATURE(23)..(23)Xaa is Lys, Glu, or
AspMISC_FEATURE(24)..(24)Xaa is Thr or Ala 22Gln Val Gln Leu Xaa
Gln Pro Gly Ala Glu Val Xaa Lys Pro Gly Ala 1 5 10 15 Ser Val Xaa
Xaa Ser Cys Xaa Xaa Ser Gly Tyr Thr Phe Thr 20
25 30 2314PRTArtificial SequenceAmino acid sequence of B4 antibody
heavy chain framework region 2 with variable amino acid residues
X3, X5, X7, and X8 (B4 VHfr2)MISC_FEATURE(3)..(3)Xaa is Lys or
ArgMISC_FEATURE(5)..(5)Xaa is Arg, Thr, or
AlaMISC_FEATURE(7)..(7)Xaa is Gly, Asp, or
GluMISC_FEATURE(8)..(8)Xaa is Gln or Lys 23Trp Val Xaa Gln Xaa Pro
Xaa Xaa Gly Leu Glu Trp Ile Gly 1 5 10 2439PRTArtificial
SequenceAmino acid sequence of B4 antibody heavy chain framework
region 3 with variable amino acid residues X6, X10, X26, X29, and
X34 (B4 VHfr3)MISC_FEATURE(6)..(6)Xaa is Lys, Asp, or
GluMISC_FEATURE(10)..(10)Xaa is Lys, Glu, or
AspMISC_FEATURE(26)..(26)Xaa is Ser, Asp, or
GluMISC_FEATURE(29)..(29)Xaa is Ser or AlaMISC_FEATURE(34)..(34)Xaa
is Val or Thr 24Tyr Asn Gln Lys Phe Xaa Gly Lys Ala Xaa Leu Thr Val
Asp Lys Ser 1 5 10 15 Ser Ser Thr Ala Tyr Met Glu Val Ser Xaa Leu
Thr Xaa Glu Asp Ser 20 25 30 Ala Xaa Tyr Tyr Cys Ala Arg 35
25104PRTHomo sapiens 25Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met
Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala
Ser Ser Gly Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro
Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu
Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly
Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp
Ala Ala Thr Tyr Tyr Cys His Gln Arg Gly Ser Tyr Thr Phe Gly 85 90
95 Gly Gly Thr Lys Leu Glu Ile Lys 100 26104PRTArtificial
SequenceAmino acid sequence of B4 antibody light chain variable
region with codons for amino acid substitutions V3A, S7E, and A54D
(B4 VKv1) 26Gln Ile Ala Leu Thr Gln Glu Pro Ala Ile Met Ser Ala Ser
Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Gly
Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Thr Ser
Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Asp Ser Gly
Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr
Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr
Tyr Tyr Cys His Gln Arg Gly Ser Tyr Thr Phe Gly 85 90 95 Gly Gly
Thr Lys Leu Glu Ile Lys 100 27104PRTArtificial SequenceAmino acid
sequence of B4 antibody light chain variable region with codons for
amino acid substitutions Q1D, I10T, M11L, and A54D (B4 VKv2) 27Asp
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Ala Ser Pro Gly 1 5 10
15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Gly Val Asn Tyr Met
20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp
Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Asp Ser Gly Val Pro Ala Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His
Gln Arg Gly Ser Tyr Thr Phe Gly 85 90 95 Gly Gly Thr Lys Leu Glu
Ile Lys 100 28104PRTArtificial SequenceAmino acid sequence of B4
antibody light chain variable region with codons for amino acid
substitutions I10T, M11L, V19A, V29A, and S75E (B4 VKv3) 28Gln Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Ala Ser Pro Gly 1 5 10 15
Glu Lys Ala Thr Met Thr Cys Ser Ala Ser Ser Gly Ala Asn Tyr Met 20
25 30 His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile
Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe
Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Glu
Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln
Arg Gly Ser Tyr Thr Phe Gly 85 90 95 Gly Gly Thr Lys Leu Glu Ile
Lys 100 29104PRTArtificial SequenceAmino acid sequence of B4
antibody light chain variable region with codons for amino acid
substitutions I10T, M11L, V19A, S51D, and L53T (B4 VKv4) 29Gln Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Ala Ser Pro Gly 1 5 10 15
Glu Lys Ala Thr Met Thr Cys Ser Ala Ser Ser Gly Val Asn Tyr Met 20
25 30 His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile
Tyr 35 40 45 Asp Thr Asp Lys Thr Ala Ser Gly Val Pro Ala Arg Phe
Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser
Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln
Arg Gly Ser Tyr Thr Phe Gly 85 90 95 Gly Gly Thr Lys Leu Glu Ile
Lys 100 30104PRTArtificial SequenceAmino acid sequence of B4
antibody light chain variable region with codons for amino acid
substitutions V3A, S7E, V19A, A54D, and S75E (B4 VKv11) 30Gln Ile
Ala Leu Thr Gln Glu Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15
Glu Lys Ala Thr Met Thr Cys Ser Ala Ser Ser Gly Val Asn Tyr Met 20
25 30 His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile
Tyr 35 40 45 Asp Thr Ser Lys Leu Asp Ser Gly Val Pro Ala Arg Phe
Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Glu
Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln
Arg Gly Ser Tyr Thr Phe Gly 85 90 95 Gly Gly Thr Lys Leu Glu Ile
Lys 100 31104PRTArtificial SequenceAmino acid sequence of B4
antibody light chain variable region with codons for amino acid
substitutions I10T, M11L, V19A, V29A, S51D, L53T, and S75E (B4
VKv34) 31Gln Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Ala Ser
Pro Gly 1 5 10 15 Glu Lys Ala Thr Met Thr Cys Ser Ala Ser Ser Gly
Ala Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Thr Ser
Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Asp Lys Thr Ala Ser Gly
Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr
Ser Leu Thr Ile Glu Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr
Tyr Tyr Cys His Gln Arg Gly Ser Tyr Thr Phe Gly 85 90 95 Gly Gly
Thr Lys Leu Glu Ile Lys 100 3223PRTArtificial SequenceAmino acid
sequence of B4 antibody light chain framework region 1 with
variable amino acid residues X1, X3, X7, X10, X11 and X19 (B4
VKfr1)MISC_FEATURE(1)..(1)Xaa is Gln or AspMISC_FEATURE(3)..(3)Xaa
is Val or AlaMISC_FEATURE(7)..(7)Xaa is Ser or
GluMISC_FEATURE(10)..(10)Xaa is Ile or ThrMISC_FEATURE(11)..(11)Xaa
is Met or LeuMISC_FEATURE(19)..(19)Xaa is Val or Ala 32Xaa Ile Xaa
Leu Thr Gln Xaa Pro Ala Xaa Xaa Ser Ala Ser Pro Gly 1 5 10 15 Glu
Lys Xaa Thr Met Thr Cys 20 337PRTArtificial SequenceAmino acid
sequence of B4 antibody light chain complementarity determining
region 2 with variable amino acid residues X3, X5, and X6 (B4
VKcdr2)misc_feature(3)..(3)Xaa can be any naturally occurring amino
acidmisc_feature(5)..(6)Xaa can be any naturally occurring amino
acid 33Asp Thr Xaa Lys Xaa Xaa Ser 1 5 349PRTArtificial
SequenceAmino acid sequence of peptides of B4 V regions predicted
to bind human HLA-DR alleles 34Val Gln Leu Gln Gln Pro Gly Ala Glu
1 5 359PRTArtificial SequenceAmino acid sequence of peptides of B4
V regions predicted to bind human HLA-DR alleles 35Val Lys Pro Gly
Ala Ser Val Arg Leu1 5 369PRTArtificial SequenceAmino acid sequence
of peptides of B4 V regions predicted to bind human HLA-DR alleles
36Val Arg Leu Ser Cys Lys Thr Ser Gly1 5 379PRTArtificial
SequenceAmino acid sequence of peptides of B4 V regions predicted
to bind human HLA-DR alleles 37Trp Val Lys Gln Arg Pro Gly Gln Gly1
5 389PRTArtificial SequenceAmino acid sequence of peptides of B4 V
regions predicted to bind human HLA-DR alleles 38Tyr Asn Gln Lys
Phe Lys Gly Lys Ala1 5 399PRTArtificial SequenceAmino acid sequence
of peptides of B4 V regions predicted to bind human HLA-DR alleles
39Phe Lys Gly Lys Ala Lys Leu Thr Val1 5 409PRTArtificial
SequenceAmino acid sequence of peptides of B4 V regions predicted
to bind human HLA-DR alleles 40Tyr Met Glu Val Ser Ser Leu Thr Ser1
5 419PRTArtificial SequenceAmino acid sequence of peptides of B4 V
regions predicted to bind human HLA-DR alleles 41Val Tyr Tyr Cys
Ala Arg Gly Ser Asn1 5 429PRTArtificial SequenceAmino acid sequence
of peptides of B4 V regions predicted to bind human HLA-DR alleles
42Ile Val Leu Thr Gln Ser Pro Ala Ile1 5 439PRTArtificial
SequenceAmino acid sequence of peptides of B4 V regions predicted
to bind human HLA-DR alleles 43Val Leu Thr Gln Ser Pro Ala Ile Met1
5 449PRTArtificial SequenceAmino acid sequence of peptides of B4 V
regions predicted to bind human HLA-DR alleles 44Val Thr Met Thr
Cys Ser Ala Ser Ser1 5 459PRTArtificial SequenceAmino acid sequence
of peptides of B4 V regions predicted to bind human HLA-DR alleles
45Val Asn Tyr Met His Trp Tyr Gln Gln1 5 469PRTArtificial
SequenceAmino acid sequence of peptides of B4 V regions predicted
to bind human HLA-DR alleles 46Trp Ile Tyr Asp Thr Ser Lys Leu Ala1
5 479PRTArtificial SequenceAmino acid sequence of peptides of B4 V
regions predicted to bind human HLA-DR alleles 47Ile Tyr Asp Thr
Ser Lys Leu Ala Ser1 5 4813DNAArtificial SequenceDescription of
Artificial SequenceArtificially Synthesized DNA Sequence
48cgtaagtgga tcc 134912DNAArtificial SequenceDescription of
Artificial SequenceArtificially Synthesized DNA Sequence
49gctagctcca gc 12
* * * * *
References